tatellano ea bokahohleng BOLELA-Seq Boikemisetso ba Boikemisetso

Litlhaloso:

• Lebitso la Sehlahisoa: TELL-SeqTM Target Enrichment
• Bakeng sa: Sebelisa Lipatlisiso Feela, eseng bakeng sa mekhoa ea ho hlahloba
• Moetsi: Universal Sequencing Technology Corporation
• E hlokehang Genomic DNA: 5ng
• Maemo a Polokelo: Tris buffer pH 7.5-8.0 kapa buffer e tlase ea TE

Litaelo tsa Tšebeliso ea Sehlahisoa

Litlhahiso tsa ho Kena ha Genomic DNA:
Netefatsa hore bonyane u na le 5ng ea genomic DNA bakeng sa protocol. DNA ea boima bo phahameng ba limolek'hule e bohlokoa bakeng sa tatellano e atlehileng. Lekanya DNA ka mokhoa o thehiloeng ho fluorometric joalo ka Qubit dsDNA BR Assay Kit kapa HS Kit. Hlakola DNA e tsepamisitsoeng ho sebetsa (0.4ng/l ho isa ho 1ng/l) ho Tris buffer pele ho tekanyo. Boloka DNA ea genomic ho Tris buffer e nang le pH 7.5-8.0 kapa buffer e tlase ea TE.

Litaba tsa Kit:

TELL-SeqTM Library Prep Kit, Standard Size e na le tse peli mabokose:

• Lebokose la 1: E na le li-reagents tse fapaneng tse kenyelletsang Barcoding Enzyme, Exonuclease, le tse ling.
• Lebokose la 2: E na le TELL Bead Plexb, Wash Solution, le Stop Solution.

Hlokomela: Se ke oa hatsetsa le ho qhibiliha Lebokose la 1 li-reagents ho feta makhetlo a 6.

LBH:

1. P: Na nka sebelisa mekhoa e meng ntle le e thehiloeng ho fluorometric ho ho lekanya DNA?
   A: Ho khothalletsoa ho sebelisa mokhoa o thehiloeng ho fluorometric joaloka Qubit dsDNA BR Assay Kit kapa HS Kit bakeng sa tekanyo e nepahetseng. Qoba mekhoa e lekanyang palo eohle ea "nucleic acid".
2. P: Ke lokela ho boloka DNA ea genomic joang bakeng sa liphetho tse nepahetseng?
   A: Genomic DNA e lokela ho bolokoa ho Tris buffer e nang le pH ho tloha ho 7.5 - 8.0 kapa TE buffer e tlase (10mM Tris-HCl, 0.1 mM EDTA, pH 8.0) bakeng sa liphello tse ntle.

Bakeng sa Tšebeliso ea Lipatlisiso Feela. Ha e sebelisoe lits'ebetsong tsa tlhahlobo.
Tokomane # 100032-USG v4.0
Phato 2024
Tokomane ena ke ea mong'a Universal Sequencing Technology Corporation 'me e reretsoe feela tšebeliso ea moreki oa eona mabapi le tšebeliso ea lihlahisoa tse hlalositsoeng mona eseng ka mabaka a mang. Litaelo tse tokomaneng ena li tlameha ho lateloa ka nepo ke basebetsi ba koetlisitsoeng hantle ho netefatsa ts'ebeliso e nepahetseng le e bolokehileng ea lisebelisoa tsa TELL-Seq™.
UNIVERSAL SEQUENCING TECHNOLOGY CORPORATION HA E AKASE MOLAMO OO EFE KA EFE KA MOR'A TSEBEDISO E fosahetseng ea TELL-SEQ™ KIT.
©2023 Universal Sequencing Technology Corporation. Litokelo tsohle li sirelelitsoe. TELL-Seq™ ke lets'oao la khoebo la Universal Sequencing Technology Corporation. Mabitso a mang kaofela, li-logo le matšoao a mang a khoebo ke thepa ea beng ba tsona.

Nalane ea Phetoho

Doc #100032-USG v1.0	La 2021 Pulungoana XNUMX	Phatlalatso ea Pele
Doc #100032-USG v2.0	Phato 2022	Ntlafatso ea protocol ho sebetsa le TELL- Seq™ Library Prep kit V1 e nang le khetho ea Suspension Buffer EZ le TELL Bead Plex.
Doc # 100032-USG v3.0	Phato 2023	E tlositsoe TELL Bead khetho. Ke feela TELL Bead Plex e sebelisoang ho ea pele. E kentse Tlhokomeliso le setšoantšo se nang le mekhoa e khothaletsoang ea ho kopanya bakeng sa mohato oa bohlokoa oa ho potoloha ha tube nakong ea ts'ebetso ea barcoding ho boloka thepa ea DNA e boima ba molek'hule.
Doc # 100032-USG v4.0	Phato 2024	Tataiso e fetotsoeng ea mosebelisi bakeng sa Kakaretso ea Boipheliso ba Maikutlo. E kentse lintlha tse ling mabapi le ho sebelisa TELL-Seq™ Library Prep le litsamaiso tse ling tsa Target Enrichment

Selelekela

Protocol ena e hlalosa mokhoa oa ho lokisa lilaebrari tsa TELL-Seq™ tse nang le indexed-indexed-end-paired ka hybridization ho sebelisoa motsoako oa TELL-Seq™ Library Prep Kit le Agilent® SureSelect Target Enrichment System. Sistimi ea TELL-Seq™ Library Prep Kit e boetse e tsamaisana le lits'ebetso tse ling tsa Maiketsetso a Maikutlo joalo ka IDT le Twist Biosciences.
TELL-Seq™ laeborari ea prep kit e sebelisa theknoloji e ncha ea Transposase Enzyme Linked Long-read Sequencing (TELL-Seq™) † ho lokisa laeborari e kopantsoeng ka bobeli ho hlahisa li-barcode tse hokahaneng ho tsoa ho sistimi ea tatellano ea Illumina®. Agilent® SureSelect Target Enrichment System e lumella ho ntlafatsa libaka tse lebisitsoeng ho sebelisa li-probes tse khethehileng haholo. Sistimi e 'ngoe le e' ngoe ea Maiketsetso ea Maiketsetso e na le sehlopha se ikhethileng sa li-probes tse lumellang hore libaka tse ikhethileng li tšoaroe tse tsamaellanang le lilaebrari tsa TELL-Seq™.

Litlhahiso tsa ho kenya Genomic DNA
5ng Genomic DNA ea hlokahala bakeng sa protocol ena. Boima bo phahameng ba molek'hule (HMW) DNA e bohlokoa bakeng sa tatellano e atlehileng ea TELL-Seq™.

• Bakeng sa genome ea motho, bonyane sampLe boholo ba DNA bo lokela ho ba kholo ho feta 40Kb.
•  HMW DNA ho tloha ho 100Kb ho isa ho 300Kb ke lisebelisoa tse nepahetseng bakeng sa ts'ebeliso e ntle ka ho fetisisa ea batho.
• Qoba ho senya HMW DNA nakong ea ho sebetsana le eona. Tlosa DNA ea boima ba molek'hule e tlase (e tsejoang e le smear e ka tlase ho 10Kb holim'a gel) ho s.ample haeba ba hlahisa karolo ea bohlokoa ho DNA sample.

Sebelisa mokhoa o thehiloeng ho fluorometric ho lekanya ho kenya DNA. Haeba u sebelisa Qubit dsDNA BR Assay Kit kapa HS Kit, sebelisa bonyane 2 µL ea DNA ka 'ngoe.ample bakeng sa tekanyo. Qoba mekhoa e lekanyang feela kakaretso ea nucleic acid, joalo ka NanoDrop kapa mekhoa e meng ea ho monya UV. Bakeng sa tekanyo e nepahetseng ea mahloriso a HMW DNA, hlapolla DNA e tsepamisitsoeng boemong ba ho sebetsa (0.4ng/µl ho isa ho 1ng/µl) ka har'a Tris buffer (pH 7.5-8.0) lihora tse 'maloa ho isa letsatsi pele ho tekanyo ea mahloriso le ho lokisa laeborari. Genomic DNA e lokela ho bolokoa ka har'a sekheo sa Tris se nang le pH ho tloha ho 7.5 - 8.0 kapa TE buffer e tlase (10mM Tris-HCl, 0.1 mM EDTA, pH 8.0). Bakeng sa ho hlahloba bohloeki ba DNA sampLe, karo-karolelano ea tekanyo ea ho monya ho 260 nm ho ea ho 280 nm e ka sebelisoa. Protocol ena e ntlafalitsoe bakeng sa DNA e nang le litekanyetso tsa ho monya tse 1.8–2.0. Haeba ho na le RNA e feteletseng ho DNA sample, e lokela ho tlosoa ka phekolo ea RNase.

Litaba tsa Kit

TELL-Seq™ Library Prep Kit, Standard Size (2 Mabokose)

Lebokose la 1 ho 2: TELL-Seq™ Library Reagent Box 1 V1 (PN 100035)

HLOKOMELA: Se ke oa hatsetsa le ho qhibilihisa li-reagents tsa Lebokose la 1 ka makhetlo a fetang 6.

a Ho sebelisoa le 10× Primer II ho TELL-Seq™ Library Multiplex Primer Kit hammoho bakeng sa laeborari amptokafatso.

Lebokose la 2 la 2: TELL-Seq™ Library Reagent Box 2 V1 (PN 100036)

Lebitso la Karolo	Mobala oa Cap	Bolumo (mL)	Mocheso oa polokelo
BOLELANG Bead Plexb	CAP Orange	76	2°C ho isa ho 8°C
Hlatsoa Tharollo	CAP White	4500	2°C ho isa ho 8°C
Emisa Tharolloc	CAP Natural	960	2°C ho isa ho 25°C

b TELL Bead Plex e sebetsa hantle ho Illumina le li-non-Illumina Sequencing Systems.
c Pele o sebelisoa, haeba Stop Solution e sa hlaka kapa e e-na le lipula tse tšoeu, futhumatsa tube ka 37C. Vortex ho qhala pula efe kapa efe. Kamora ts'ebeliso ea pele, boloka Stop Solution e tsosolositsoe mocheso oa kamore bakeng sa tšebeliso ea nako e tlang.

TLHOKOMELO

 TELL-Read pipeline v1.1 kapa ka holimo e ea hlokahala ho sekaseka tatellano ea data e hlahisitsoeng lilaebraring tsa TELL-Seq™ tse lokiselitsoeng ka TELL Bead Plex.

TELL-Seq™ Library Prep Kit, HT24 (2 Mabokose)
Lebokose la 1 ho tse 2: TELL-Seq™ Library Reagent Box 1 V1, HT24 (PN 100037) 

HLOKOMELA: Se ke oa hatsetsa le ho qhibilihisa li-reagents tsa Lebokose la 1 ka makhetlo a fetang 6. 

a Ho sebelisoa le 10× Primer II ho TELL-Seq™ Library Multiplex Primer Kit hammoho bakeng sa laeborari amptokafatso.

Lebokose la 2 ho tse 2: TELL-Seq™ Library Reagent Box 2 V1, HT24 (PN 100038)

Lebitso la Karolo	Mobala oa Cap		Bolumo	Mocheso oa polokelo
BOLELANG Bead Plexb	namunu		456 mL	2°C ho isa ho 8°C
Hlatsoa Tharollo		Boputsoa	28.5 mL	2°C ho isa ho 8°C
Emisa Tharolloc		Bosoeu	5.76 mL	2°C ho isa ho 25°C

b TELL Bead Plex e sebetsa hantle ho Illumina le li-non-Illumina Sequencing Systems.
c Pele o sebelisoa, haeba Stop Solution e sa hlaka kapa e na le pula e tšoeu, futhumatsa tšupu ho 37 ºC. Vortex ho qhala pula efe kapa efe. Kamora ts'ebeliso ea pele, boloka Stop Solution e tsosolositsoe mocheso oa kamore bakeng sa tšebeliso ea nako e tlang.

KELETSO EA PRO: PEDI YA TELL-Seq™ Library Prep Kits, HT24 ho kenyeletswa Box 1 le Box 2 ka bobedi di ka kopanngwa le TELL-Seq™ Library efe kapa efe ya Multiplex Primer Kits.

TLHOKOMELO 
TELL-Read pipeline v1.1 kapa ka holimo e ea hlokahala ho sekaseka tatellano ea data e hlahisitsoeng lilaebraring tsa TELL-Seq™ tse lokiselitsoeng ka TELL Bead Plex.

TELL-Seq™ Library Multiplex Primer (1-8) Kit (PN 100003)

KELETSO EA PRO: One TELL-Seq™ Library Multiplex Primer (1-8) Kit e na le li-primers tse lekaneng hore li ka sebelisoa le FOUR TELL-Seq™ WGS Library Prep Kits.

TELL-Seq™ Library Multiplex Primer (9-16) Kit (PN 100009)

KELETSO EA PRO: ONE TELL-Seq™ Library Multiplex Primer (9-16) Kit e na le li-primers tse lekaneng hore li ka sebelisoa le FOUR TELL-Seq™ WGS Library Prep Kits, Standard Size.

TELL-Seq™ Library Multiplex Primer (17-24) Kit (PN 100010)

KELETSO EA PRO: ONE TELL-Seq™ Library Multiplex Primer (17-24) Kit e na le li-primers tse lekaneng hore li ka sebelisoa le FOUR TELL-Seq™ WGS Library Prep Kits, Standard Size.

TELL-Seq™ Illumina® Sequencing Primer Kit (PN 100004)

Lebitso la Karolo	Mobala oa Cap	Ho tsepamisa maikutlo	Bolumo (mL)	Mocheso oa polokelo
Bala 1 Primer	Ntsho	100mM	50	-25°C ho ea ho -15°C

Bala 2 Primer	Bosoeu	100mM	50	-25°C ho ea ho -15°C
Index 1 Primer	Khubedu	100mM	50	-25°C ho ea ho -15°C
Index 2 Primer	Bosehla	100mM	50	-25°C ho ea ho -15°C

TELL-Seq™ Target Blocker (PN 100019)

Lebitso la Karolo	Mobala oa Cap	Bolumo (mL)	Mocheso oa polokelo
TELL-Seq™ Target Blocker	CAP White	40	-25°C ho ea ho -15°C

Lisebelisoa le Lisebelisoa (ha li fanoe)

Lintho tse ka sebelisoang

Consumable	Mofani
0.2 mL ea PCR kapa tube e hlobolang	Kakaretso lab mofani wa thepa
20 mL pipette ntlha (e tloaelehileng le e pharaletseng e orifice)	Kakaretso lab mofani wa thepa
200 mL pipette ntlha (e tloaelehileng le e pharaletseng e orifice)	Kakaretso lab mofani wa thepa
Bopaki ba Ethanol 200 (bo feletseng) bakeng sa baeloji ea limolek'hule (500 mL)	Sigma-Aldrich, # E7023
Metsi a se nang metsi	Kakaretso lab mofani wa thepa
AMPke XP	Beckman, # A63880
Agilent Bioanalyzer High Sensitivity DNA Analysis Kit*	Agilent, # 5067-4626
TapeStation High Sensitivity D5000 ScreenTape Assay*	Agilent, # 5067-5592, #5067-5593
Qubit dsDNA HS Assay Kit	Thermo Fisher Scientific, # Q32851 kapa Q32854
Qubit Assay Tubes	Thermo Fisher Scientific, # Q32856
Dynabeads MyOne Streptavidin T1	Thermo Fisher Scientific, # 65601, 65602 kapa 65603
Agilent SureSelect XT HS Capture Library Human All Exon 7	Agilent, # G9704N, G9705N kapa G9706N
Agilent SureSelect XT HS Target Enrichment Kit, ILM Hyb Module (Post PCR), 16 Rxn	Agilent, #G9916B
TE buffer, pH 8.0	Kakaretso lab mofani wa thepa

* Ho ipapisitse le sistimi e fumanehang sebakeng sa basebelisi.

Thepa

Thepa	Mofani
Thermo Cycler	Li-Biosystem tse sebelisitsoeng
Magnetic ema bakeng sa 0.2 mL PCR li-tubes	Kakaretso lab mofani wa thepa
Rotator ea tube	Kakaretso lab mofani wa thepa
Incubator (bakeng sa 35°C)	Kakaretso lab mofani wa thepa
Vortexer	Kakaretso lab mofani wa thepa
Microcentrifuge	Kakaretso lab mofani wa thepa
Agilent Bioanalyzer*	Agilent
Agilent TapeStation*	Agilent
Qubit® Fluorometer 3.0	Thermo Fisher Scientific, # Q33216, Q33217 kapa Q33218
Leqhoa Bucket	Kakaretso lab mofani wa thepa
SpeedVac Vacuum Concentrators (Ha ho boikhethelo)	Thermo Fisher Scientific

* Ho ipapisitse le sistimi e fumanehang sebakeng sa basebelisi.

TELL-Seq™ Human Exom Capture Workflow

TELL-Seq™ WGS Library Prep

Protocol

TELL-Seq™ WGS Library Prep
Protocol e latelang e hlalosa ts'ebetso e fetotsoeng ea TELL-Seq™ eohle ea genome ea ho hlophisa laebrari ka TELL-Seq™ Library Prep kit e sebelisang DNA ea motho.amples. Mefuta e fapaneng ea liphatsa tsa lefutso tsa DNAample mefuta le eona e ka nkeloa sebaka ho latela protocol e tšoanang. Lilaebrari tsa TELL-Seq™ li tsamaisana le lits'ebetso tse ling tsa Target Enrichment, empa Agilent SureSelectXT HS Target Enrichment System e sebelisang SureSelect Human All Exon V8 capture probes e sebelisoa e le ex.ample ka protocol. Bakeng sa ts'ebeliso ho Lits'ebetso tse ling tsa Target Enrichment ka kopo latela protocol ea TELL-Seq™ Library Prep (leq. 10-22) ho hlahisa lilaeborari tse nepahetseng. Lilaebrari tsa TELL-Seq™ li ka sebelisoa e le Kenyeletso ea DNA ho latela protocol ea Sistimi e 'ngoe le e 'ngoe ea Maiketsetso ea Target hammoho le TELL-Seq™ Target Blockers e sebelisoang ho kenyelletsa li-blockers tse boletsoeng.

DNA ea barcode

Lintho tse ka sebelisoang
➢ Kenya genomic DNA (Mosebelisi)

Boholo ba Genome	Chelete e Kenyelelitsoeng	Rection Vol (mL)	Preps/Kit
E kholo (Motho)	5 ng	150	4

HLOKOMELA: 

1. Genomic DNA e lokela ho bolokoa le ho hlapolloa ka har'a "tris buffer" e nang le pH ho tloha ho 7.5 ho isa ho 8.0 kapa TE buffer e tlase (10mM Tris-HCl, 0.1 mM EDTA, pH 8.0).

➢ 5× Reaction Buffer (Kit Box 1, Boputsoa)
➢ Cofactor II (Kit Box 1, Amber)
➢ Enzyme ea Barcoding (Kit Box 1, Ntsho)
➢ BOLELANG Bead kapa TELL Bead Plex (Kit Box 2, Namunu)
➢ Suspension Buffer EZ (Kit Box 1, Tlhaho)
➢Metsi a se nang nyutlelie (Mosebelisi)
➢ 0.2 mL PCR tube kapa strip tube (Mosebelisi)
➢20 µL le 200 µL malebela a pipette e pharalletseng (Mosebelisi)

Tokisetso

1. Lokisetsa lisebelisoa tse latelang:
   

   Ntho	Polokelo	Taelo
   5 × Reaction Buffer	-25°C ho ea ho -15°C	Qhibiliha mocheso oa kamore. Vortex ho kopanya, joale   centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa.
   Cofactor II	-25°C ho ea ho -15°C	Vortex ho kopanya, ebe centrifuge ka bokhutšoanyane. Boloka ho mocheso oa kamore lefifing. Koala tube   sekoahelo ka thata ka mor'a tšebeliso e 'ngoe le e' ngoe.
   Enzyme ea Barcoding	-25°C ho ea ho -15°C	Flick tube ka makhetlo a 4 ho isa ho a 5 ho kopanya. Centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa.
   BOLELANG Bead Plex	2°C ho isa ho 8°C	Centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa. Koala sekoahelo sa tube  ka thata ka mor'a tšebeliso e 'ngoe le e' ngoe ho qoba moya ofe kapa ofe.
   Sekhahla sa ho Emisa EZ	-25°C ho ea ho -15°C	Qhibiliha mocheso oa kamore. Vortex ho kopanya, ebe centrifuge ka bokhutšoanyane. Boloka ho mocheso oa kamore.
   Metsi a se nang metsi	Mocheso oa Kamore	Boloka mocheso oa kamore.

2. Hlophisa tube rotator ka incubator ea 35 °C (sheba Mohato oa 7 oa karolo ea Tsamaiso).

TLHOKOMELO
Sebelisa malebela a pharaletseng a orifice pipette ho fetisetsa le ho kopanya limolek'hule tse phahameng tsa genomic DNA ho qoba ho senya DNA. Haeba malebela a sephara a orifice pipette ha a fumanehe, khaola 2mm-3mm ho tloha ntlha e tloaelehileng ea pipette ka lehare le hloekileng pele o sebelisoa.

Tsamaiso

1. Vortex TELL Bead Plex ka matla bonyane metsotsoana e 30. Pulse spin (centrifuge e sa feteng motsotso o le mong) ho theola tharollo ea lifaha e teng holim'a sekoahelo kapa mahlakoreng a tube. Hang pele o sebelisoa, penya TELL Bead Plex ka ntlha ea 1 µL holimo le tlase makhetlo a 200 ho etsa bonnete ba hore lifaha tsohle li emisitsoe hantle.
2. Ka tube ea 0.2 mL ea PCR, kopanya karabo ka 'ngoe ka tatellano e latelang.
   

   Reagent	Bolumo ho latela karabelo (mL)
   	Genome e kholo (150 mL)

   5 × Reaction Buffer	30
   Metsi a se nang metsi Cofactor II	20 - Z (Z ke karolo ea DNA)30
   BOLELANG Bead Plex (0.5M libarcode/mL)	19

3. Kopanya hantle ka ho kenya lipeipi holimo le tlase makhetlo a 10 kapa ho vortexing ka matla metsotsoana e 5 le pulse spin ho theola tharollo tlase. Kenya Barcoding Enzyme.
   

   Reagent	Bolumo ho latela karabelo (mL)
   	Genome e kholo
   Enzyme ea Barcoding	6 mL

4. Kopanya hantle ka ho kenya liphaephe holimo le tlase ka makhetlo a 8. Qoba ho kenya li-bubble tsa moea ha u ntse u phunya pipette ka ho boloka ntlha ea pipette ka tlaase ho tharollo ka har'a tube.
5. Sebelisa ntlha e pharaletseng ea pipette ea orifice, eketsa reagent e latelang.
   HLOKOMELA: Suspension Buffer EZ e na le viscous haholo. E-ba hlokolosi le pipette butle ho netefatsa hore molumo o nepahetseng o fanoa.
6. Beha molumo oa pipette ho 110 μL. Sebelisa ntlha e pharaletseng ea orifice pipette, ka bonolo kopanya tharollo ka ho penya butle-butle holimo le tlase makhetlo a 6-8. Qoba ho hlahisa li-bubble tse ngata tsa moea ha u le pipette ka ho boloka ntlha ea pipette ka tlaase ho tharollo ka har'a tube.
7. Beha sample tube holim'a tube rotator ka 35 ° C incubator 'me u potoloha butle (10-15 rpm) metsotso e 30.

Sample li-tubes tse behiloeng holim'a Tube Rotator ka har'a incubator ea 35 ° C.

Hlokomela: Phetoho e nepahetseng ea li-tube e bohlokoa ho baballa thepa ea HMW DNA le ho tsamaisa mokhoa o nepahetseng oa barcoding. Sistimi ea ho kopanya e khothalelitsoeng e bonts'itsoe ka tlase (lehlakoreng le letšehali). Litsamaiso tse kopanyang tse sa potolohang kapa tse hlahisang ho sisinyeha ka matla ha li lumellane le ho boloka thepa ea HMW DNA le TELL-Seq; tse ling tsa litsamaiso tsena li boetse li bontšoa ka tlase (lehlakoreng le letona).

Tsitsisa DNA 

Lintho tse ka sebelisoang
➢ Stabilizer (Kit Box 1, Violet)

Tokisetso

1. Lokisetsa lisebelisoa tse latelang:

Ntho	Polokelo	Taelo
Setsibotsi	-25°C ho ea ho -15°C	Flick tube ka makhetlo a 4 ho isa ho a 5 ho kopanya. Centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa.

Tsamaiso

1. Khutlisa sample tube ho tloha ho incubator ea 35 ° C.
2. Kenya Stabilizer ka har'a tube.
   

   Reagent	Bolumo ho latela karabelo (mL)
   	Genome e kholo
   Setsibotsi	3

3. Beha molumo oa pipette ho 110 μL. Sebelisa ntlha e pharaletseng ea orifice pipette, ka bonolo kopanya tharollo ka ho penya butle-butle holimo le tlase makhetlo a 6-8. Qoba ho etsa li-bubble tse ngata.
4. Beha samp'me u khutlele holim'a rotator ea tube ka har'a incubator ea 35 ° C 'me u e fetole butle (10-15 rpm) metsotso e 30.

Taghlahisa DNA 

Lintho tse ka sebelisoang
➢ TagEnzyme ea ho hola (Kit Lebokose la 1, Bokhubelu)
➢ Exonuclease (Kit Box 1, Yehla)

Tokisetso

1. Lokisetsa lisebelisoa tse latelang:
   

   Ntho	Polokelo	Taelo
   Tagenzyme	-25°C ho ea ho -15°C	Flick tube ka makhetlo a 4 ho isa ho a 5 ho kopanya. Centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa.
   Exonuclease	-25°C ho ea ho -15°C	Flick tube ka makhetlo a 4 ho isa ho a 5 ho kopanya. Centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa.

2. Sebelisa rotator ea tube e tšoanang ho 35 °C incubator.

Tsamaiso

1. Khutlisa sample tube ho tloha ho incubator ea 35 ° C.
2. Eketsa TagHo ruruha ha Enzyme le Exonuclease ka har'a tube.
   

   Reagent	Bolumo ho latela karabelo (mL)
   	Genome e kholo
   Tagenzyme	2
   Exonuclease	3

3. Beha molumo oa pipette ho 110 μL. Sebelisa ntlha e pharaletseng ea pipette ea orifice, kopanya tharollo ka bonolo butle-butle ho ea holimo le tlase ka makhetlo a 8. Bakeng sa mokhoa ona, ho kopanya ho lokela ho ba bonolo haholo. Qoba ho etsa li-bubble tse ngata.
4. Beha sampkhutlela ho tube rotator ka 35 ° C e incubator 'me u e fetole butle metsotso e 10. Ha ho hlokahala, palo e fapaneng ea TagEnzyme e ka sebelisoa ho fetola boholo ba laebrari.
   HLOKOMELA: Haeba ho ratoa ho kenya laeborari e telele, ho fokotsoe palo ea Tagging Enzyme e ka sebelisoa karabelong. Ka lehlakoreng le leng, haeba ho khethoa laeborari e khuts'oane ea ho kenya, ho fihla ho 6µL Tagging Li-enzyme li ka sebelisoa karabelong.
5. Tsoela pele mohatong o latelang hang ka mor'a ho qhoqhoa.

Hlatsoa Lifaha

Lintho tse ka sebelisoang

• Emisa Tharollo (Kit Box 2, Tlhaho kapa e bolokiloe mocheso oa kamore ka mor'a tšebeliso ea pele)
• Hlatsoa Tharollo (Kit Box 2, Mosoeu)
• 0.2 mL PCR tube kapa strip tube (Mosebelisi)

Tokisetso

1. Lokisetsa lisebelisoa tse latelang:
   

   Ntho	Polokelo	Taelo
   Emisa Tharollo	2°C ho isa ho 25°C	Sheba hore na ho na le lipula life kapa life. Ha e le teng, futhumala buffer ho 37°C metsotso e 10, le vortex ho fihlela e qhibiliha. Boloka mocheso oa kamore bakeng sa  tshebediso ya nako e tlang.
   Hlatsoa Tharollo	2°C ho isa ho 8°C	Tlisa mocheso oa kamore.

2. Theha sebaesekele sa thermo ka lenaneo le latelang:
   • Khetho ea sekoaelo sa preheat ho 100°C
   • 63°C ka ho sa feleng

Tsamaiso

1. Beha sample tube holim'a sethala sa makenete bakeng sa motsotso o le 1 kapa ho fihlela tharollo e hlakile.
2. Ha tube e ntse e le holim'a magnetic stand, aspirate 'me u lahle supernatant ntle le lifaha tse senyang.
3. Tlosa tube ho tloha sethaleng sa makenete. Kenya 120 µL Hlatsoa Tharollo ho sample tube. Pipet ho emisa lifaha hape. Haeba ho hlokahala, pulse spin ho theola tharollo.
4. Beha sampLe tube e khutlele holim'a sethala sa makenete bakeng sa motsotso o le 1 kapa ho fihlela tharollo e hlakile.
5. Ha tube e ntse e le holim'a magnetic stand, aspirate 'me u lahle supernatant ntle le lifaha tse senyang.
6. Tlosa tube ho tloha sethaleng sa makenete. Kenya 80 µL ea Stop Solution ho tube.
7. Pipe ka makhetlo a 'maloa ho emisa lifaha hape. Haeba ho hlokahala, pulse spin ho theola tharollo.
8. Eketsa tube ka mocheso oa kamore ka metsotso e 5.
9. Beha sampLe tube e khutlele holim'a sethala sa makenete bakeng sa motsotso o le 1 kapa ho fihlela tharollo e hlakile.
10. Ha tube e ntse e le holim'a magnetic stand, aspirate 'me u lahle supernatant ntle le lifaha tse senyang.
11. Tlosa tube ho tloha sethaleng sa makenete. Kenya Tharollo ea ho Hlatsoa ea 120 µL ho tube ea PCR. Pipet ho emisa lifaha hape.
12. Fetisetsa tharollo ea lifaha ka har'a tube e ncha ea 0.2ml PCR.
13. Eketsa tube ka 63 °C ho PCR thermocycler ka metsotso e 3.
14. Beha e ncha sample tube holim'a sethala sa makenete mocheso oa kamore bakeng sa motsotso o le mong kapa ho fihlela tharollo e hlakile.
15. Ha tube e ntse e le holim'a magnetic stand, aspirate 'me u lahle supernatant ntle le lifaha tse senyang.
16. Tlosa tube ho tloha sethaleng sa makenete. Kenya Tharollo ea ho Hlatsoa ea 120 µL ho tube ea PCR. Pipet ho emisa lifaha hape. Haeba ho hlokahala, pulse spin ho theola tharollo.
17. Eketsa tube ka 63 °C ho PCR thermocycler ka metsotso e 3.
18. Beha sample tube holim'a sethala sa makenete mocheso oa kamore bakeng sa motsotso o le mong kapa ho fihlela tharollo e hlakile.
19. Ha tube e ntse e le sethaleng sa makenete, lakatsa 'me u lahle matla a phahametseng tlhaho ntle le ho senya lifaha. Sebelisa pipette ea P20 ho tlosa supernatant leha e le efe e setseng.
20. Tlosa tube ho tloha sethaleng sa makenete. Emisa lifaha hape ka 20 µL ea Wash Solution.

HLOKOMELA
Ena ke TLHOKOMELISO EA MAEMO. Lifaha tse hlatsoitsoeng li ka bolokoa ho 2°C ho isa ho 8°C libeke tse peli.

AmpLify Library

Lintho tse ka sebelisoang

• 2 × PCR Master Mix (Kit Box 1, Pinki)
• 10 × Primer I (Kit Box 1,  Mosoeu)
• 10× Primer II, T50# (Multiplex Primer Kit)
• Metsi a se nang nyutlelie (Mosebelisi)
• 0.2 mL PCR tube kapa strip tube (Mosebelisi)

Tokisetso

1. Lokisetsa lisebelisoa tse latelang:
   

   Ntho	Polokelo	Taelo
   2 × PCR Master Mix	-25°C ho ea ho -15°C	Qhibiliha mocheso oa kamore. Beha tube 4 ho ea ho 5  linako tsa ho kopanya, ebe centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa.
   10 × Primer I	-25°C ho ea ho -15°C	Qhibiliha mocheso oa kamore. Vortex ho kopanya, joale centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa.
   10× Primer II, T50#	-25°C ho ea ho -15°C	Qhibiliha mocheso oa kamore. Vortex ho kopanya, joale  centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa.
   Morekisi	-25°C ho ea ho -15°C	Qhibiliha mocheso oa kamore. Vortex ho kopanya, joale centrifuge ka bokhutšoanyane. Boloka mocheso oa kamore.
   Metsi a se nang metsi	Mocheso oa Kamore	Boloka mocheso oa kamore.

2. Beha Library Amplification Program (LAP) ho sebaesekele sa thermo ka tsela e latelang:
   • 63°C metsotso e 2
   • 72°C metsotso e 2
   • 98°C metsotsoana e 30
   • [98°C metsotsoana e 15, 63°C metsotsoana e 20, 72°C metsotsoana e 30] x Cycle Number
   • 72°C metsotso e 3
   • 4°C ka ho sa feleng

HLOKOMELA:
Nomoro ea potoloho e feto-fetoha ho ipapisitsoe le lits'ebetso le litlhoko tse tlase. Khothalletsa ho qala ka li-cycles tse 13. Nomoro e phahameng ea potoloho e tla hlahisa laebrari e ngata ea TELL-Seq e le kenyelletso bakeng sa Hybridization le Capture, empa litefiso tse phahameng tsa ho pheta-pheta bakeng sa tlhahlobo ea ho qetela ea tatellano. Nomoro e tlase ea potoloho e ka fokotsa sekhahla sa ho pheta-pheta, empa lintlha tse tlase tsa DNA li ka baka ho fokotseha ha bokhoni ba ho hapa le ho fokotsa ho rarahana ha laeborari. Ka kopo sheba pheletso ea Qualify and Quantify Library for Hybridization and Capture bakeng sa lintlha tse ling ha ho khethoa nomoro ea potoloho e nepahetseng.

Boholo ba Genome	Bolulo ba Lifaha tse Sebelisitsoeng (B) bakeng sa PCR	Bophahamo ba PCR	Nomoro ea potoloho
E kholoanyane	20 mL	75 mL	12-14

Tsamaiso

1. Lifaha tsa Vortex ka matla bakeng sa metsotsoana e 10 ho emisa lifaha hape. Pulse spin ho theola tharollo. U sebelisa ntlha ea 20 µL, penya lifaha holimo le tlase makhetlo a 5 ho etsa bonnete ba hore lifaha tsohle li fanyehiloe hantle. Hang-hang fetisetsa boholo ba tharollo ea lifaha ho tube e ncha ea PCR.
2. Beha tube ea PCR sebakeng sa makenete bakeng sa motsotso oa 1 kapa ho fihlela tharollo e hlakile.
3. Ha tube e le sebakeng sa makenete, tlosa 20 µ L supernatant ntle le lifaha tse sitisang. Tlosa tube ea PCR ho makenete.
4. Kenya li-reagents tse latelang ho tube ea PCR.
   

   Reagent	Bolumo ho latela karabelo (µL)
   	Genome e kholo (75 μL)
   Metsi a se nang metsi	16 mL
   2 × PCR Master Mix	37.5 mL
   10 × Primer I	7.5 mL
   10× Primer II, T50#	7.5 mL
   Morekisi Botala	4.5 mL

5. Kopanya hantle ka vortexing kapa pipetting. Pulse spin ho theola tharollo.
6. Beha tube holim'a sebaesekele sa mocheso 'me u tsamaise lenaneo la LAP (sheba ka holimo) ka palo e nepahetseng ea lipotoloho.
7. Ka mor'a PCR amplification, sebelisa sehlahisoa sa 2 µL PCR bakeng sa tlhahlobo ea boleng ho Bioanalyzer kapa TapeStation. Sheba karolo ea Qualify and Quantify Library bakeng sa litaelo.

KELETSO EA PRO: Haeba tlhahlobo ea QC e bontša hore lihlahisoa tsa laebrari li batla li le tlaase, beha tube e nang le sehlahisoa sa PCR se setseng ho thermocycler le ampfana ka potoloho e le 'ngoe kapa tse peli pele u fallela karolong ea ho Hloekisa Laebrari.

HLOKOMELA:
Ena ke TLHOKOMELISO EA MAEMO. Sehlahisoa sa PCR se ka bolokoa ho -25°C ho ea ho -15°C khoeli e le 'ngoe.

Hloekisa Laebrari

Lintho tse ka sebelisoang

• AMPure XP (Mosebelisi)
• Bopaki ba Ethanol 200 (bo felletseng) bakeng sa baeloji ea limolek'hule (Mosebelisi)
• Metsi a se nang nyutlelie (Mosebelisi)
• 0.2 mL PCR tube kapa strip tube (Mosebelisi)

Tokisetso

1. Lokisetsa lisebelisoa tse latelang:

Ntho	Polokelo	Taelo
Fresh 75% (v/v) ethanol	Mocheso oa Kamore	E hloka 400 ml ka motsotsoanaample. Kopanya 1.5 mL Ethanol (bopaki ba 200) le 0.5 mL metsi a se nang Nuclease. Vortex ho kopanya le ho boloka mocheso oa kamore.
AMPke XP	2°C ho isa ho 8°C	E tlise mocheso oa kamore bonyane metsotso e 20 le vortex ka matla ho emisa lifaha pele li sebelisoa.
Metsi a se nang metsi	Mocheso oa Kamore	Boloka mocheso oa kamore.
TE buffer, pH 8.0	Mocheso oa Kamore	Boloka mocheso oa kamore.

Tsamaiso

1. Ka bokhutšoanyane centrifuge sample PCR tube ho theola tharollo eohle.
2. Beha tube holim'a sethala sa makenete bakeng sa motsotso o le 1 kapa ho fihlela tharollo e hlakile.
3. Ha tube e ntse e le sethaleng sa makenete, fetisetsa supernatant ho tube e ncha ea 0.2 mL PCR ntle le lifaha tse khathatsang.
4. Lekanya molumo oa supernatant e fetisitsoeng (sehlahisoa sa PCR) ka pipette.
5. Kenya li-reagents tse latelang sehlahisoa sa PCR ho kakaretso ea 100 µL.
   

   Reagent	Moqolo ka karabelo
   PCR sehlahisoa	75 mL
   Metsi a se nang metsi	Ho fihlela kakaretso ea 100 ml

6. Vortex ka matla ho emisa hape ho AMPure XP mme o kenye 78 μL AMPu kenya XP sehlahisoa sa 100 µL PCR.
7. Kopanya ka liphaephe holimo le tlase makhetlo a 10.
8. E kenye ka mocheso oa kamore ka metsotso e 5.
9. Beha tube holim'a sethala sa makenete bakeng sa motsotso o le 1 kapa ho fihlela tharollo e hlakile.
10. Aspirate le lahla supernatant ntle le ho khathatsa AMPure lifaha.
11. Ha u ntse u boloka pompo e eme sebakeng sa makenete, eketsa 200 µL e sa tsoa lokisoa e 75% ea ethanol ka har'a tube. E lumelle ho lula metsotsoana e 30.
12. Aspirate le lahla matla a phahametseng tlhaho ntle le lifaha tse sitisang.
13. Pheta mehato ea 11-12 nako e 'ngoe hape, u boloke tube e eme ka matla a khoheli nako eohle.
14. Boloka tšupu e holim'a sethala sa makenete e butse kepisi 'me u lumelle tšupu hore e ome ka metsotso e 1-2 ho etsa mouoane mesaletsa ea ethanol. U SE KE UA omisa lifaha ho feta tekano.
15. Tlosa tšupu sebakeng sa makenete 'me u kenye metsi a se nang li-nuclease tse 20 µL ho lifaha.
16. Pipette kapa vortex ho emisa lifaha hape. E lumelle ho lula metsotso e 5.
17. Beha tube holim'a sethala sa makenete bakeng sa motsotso o le 1 kapa ho fihlela tharollo e hlakile.
18. Fumana 18 µL ea matla a holimo ho tube e ncha. Hlokomela hore u se ke ua senya lifaha.
19. Matla a holimo a na le laeborari ea TELL-Seq™.

HLOKOMELA:
Ena ke NTHO E BOLOKEHILENG EA MAEMO. Laeborari e hloekisitsoeng ea TELL-Seq e ka bolokoa ho -25°C ho ea ho -15°C khoeli kaofela.

Tšoaneleha 'me U Lekanye Laebrari bakeng sa Boikemisetso ba Boipheliso

Lintho tse ka sebelisoang

• Agilent High Sensitivity DNA Kit kapa TapeStation High Sensitivity D5000 ScreenTape Assay (Mosebelisi)
• Qubit dsDNA HS Assay Kit (Mosebelisi)
• TE buffer, pH 8.0 (Mosebelisi)

HLOKOMELA:
Tekolo e tloaelehileng ea laeborari ea qPCR bakeng sa sistimi ea Illumina e sebetsa bakeng sa laeborari ea TELL-Seq, empa ha e hlokehe.

Tokisetso

1. Lokisetsa lisebelisoa tse hlokahalang ho latela Bioanalyzer kapa TapeStation le Qubit.

Tsamaiso

1. Sebelisa 1 µL ea laebrari bakeng sa Agilent High Sensitivity DNA Kit kapa 2 µL ea laeborari bakeng sa TapeStation High Sensitivity D5000 ScreenTape Assay.
2. Sheba sehlahisoa sa PCR se bolokiloeng se sa hloekang ho tsoa ho Amplify Library karolo ka nako e tšoanang. Sehlahisoa sa PCR se sa hloekang se ka ba le boemo bo phahameng ba primer dimer le adapter dimer. E hloka ho hlapolloa ka makhetlo a mabeli ka metsi a se nang nuclease pele e kenya chip ea Bioanalyzer kapa theipi ea TapeStation ho qoba ho kena-kenana le lets'oao le tlase.
3. Laeborari ea boholo bo botle e lokela ho ba le likhechana tse ngata tsa laebrari ka tlase ho 1000 bp (Setšoantšo sa 1).Setšoantšo sa 1. Example ea hloekisitsoeng laeborari profile ho tsoa tekong ea TapeStation High Sensitivity D5000 Screen Tape.
4. Laeborari e ka bolokoa ho -25°C ho ea ho -15°C.
5. Etsa laeborari ea TELL-Seq™ e hlapolotsoeng ka makhetlo a 10ample: hlapolla 2 µL ea laeborari ea TELL-Seq™ ka 18 µL ea metsi a se nang nuclease. Sebelisa laeborari e hlapolotsoeng ea 4 µL ho lekola khatello ea maikutlo ka Qubit dsDNA HS Assay Kit.
6. Sebelisa mahloriso (ng/µL) le molumo ho bala kakaretso ea boima ba Laeborari e 'ngoe le e 'ngoe ea TELL-Seq™ e kenang ts'ebetsong ea SureSelectXT HS Target Enrichment System. 500 ng -1,000 ng TELL-Seq™ laebrari ka letsampLe e khothaletsoa bakeng sa liphetho tse nepahetseng, empa likenyelletso tsa laeborari li tlase joalo ka 250 ng ka sampli ka etsahala le hoja ts'ebetso e ka ba le tšusumetso e mpe.
   HLOKOMELA:
   Ho na le lithibelo tsa molumo bakeng sa protocol ea Hybridization le Capture. 12 µL ke bophahamo ba bolumo bo lumelletsoeng ho kenya DNA. Ho lokela ho nahanoa ka hloko ho etsa bonnete ba hore bolumo ea laebrari ea DNA e oela ka har'a mofuta ona. Ka mokhoa o nepahetseng sebelisa lisebelisoa tsohle tsa laebrari tsa TELL-Seq kamora QC ho Hybridization le Capture reaction.
7. (Ka boikhethelo) lilaebrari tsa TELL-Seq™ li ka tsepamisoa ka ho sebelisa SpeedVac Vacuum Concentrator. Haeba SpeedVac e tla sebelisoa bakeng sa ho tsepamisa maikutlo laebraring, TELL-Seq™ laeborari kamora moo AMPHo hloekisa XP ho ka tlosoa lifaha tsa XP ka bonyane metsi a se nang nuclease a 30 µL bakeng sa ho hlaphoheloa hamolemo. Kamora QC, laeborari e hloekileng ea TELL-Seq™ e ka tsepamisoa ho bophahamo bo lakatsehang bakeng sa Hybridization le Capture.

HLOKOMELA:
Lits'ebetso tse ling tsa ntlafatso ea sepheo li ka sebelisoa joalo ka IDT le Twist Biosciences ho sebelisa lilaebrari tsa TELL-Seq™ tse entsoeng. Latela liprothokholo tse boletsoeng tsa Morero oa Matlafatso u sebelisa lilaebrari tsa TELL-Seq™ joalo ka Kenyeletso ea DNA. Sebelisa 5ul ea TELL-Seq™ TargetSeq Blocker hammoho le li-blocker tse qaqileng ho protocol e 'ngoe le e 'ngoe.

SureSelect Target Ntlafatso
Protocol e latelang ke phetoho ea karolo ea Hybridization le Capture SureSelect XT HS Target Enrichment System bakeng sa Illumina Paired-End Multiplexed Sequencing Library Protocol (Agilent, G9702-90000). Liphetoho li lumella ho lumellana ha TELL-Seq™ WGS Library Prep le Agilent SureSelect XT HS Target Enrichment System le All Exon V8 Capture Probes. Li-reagents tsa Agilent bakeng sa Hybridization le Capture feela li ka rekoa ka thoko ka sebopeho sa 16-reaction (Agilent Part Number G9916B). Haeba u sebelisa Litsamaiso tse ling tsa Maiketsetso a Target latela liprothokholo tse boletsoeng bakeng sa sistimi ka 'ngoe le ho beha lilaebrari tsa TELL-Seq™ joalo ka DNA Input. 5ul ea TELL-Seq™ Target Blockers e boetse ea hlokahala ho kenyelletsa li-blocker tse qaqileng ho protocol e 'ngoe le e 'ngoe ea Maiketsetso a Sepheo.

Hybridize TELL-Seq™ Library ho Phanele ea Capture ea Exome

Lintho tse ka sebelisoang

• SureSelect XT HS le XT Low Input Blocker Mix, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit, ILM Hyb Module, Box 2 (Post PCR), Boputsoa)
• SureSelect RNase Block, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 2 (Post PCR), Violet)
• SureSelect Fast Hybridization Buffer, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 2 (Post PCR), Bottle)
• TELL-Seq™ Target Blocker (UST, TELL-Seq™ Target Blocker Lebokose, Mosoeu)
• SSel XT HS le XT Low Input Human All Exon V7 (Agilent, SSel XT HS le XT Low Input Human All Exon V7, Bokhubelu)
• Metsi a se nang nyutlelie (Mosebelisi)
• 0.2 mL PCR tube kapa strip tube (Mosebelisi)
• TELL-Seq™ Library (Mosebelisi)

Chelete e Kenyelelitsoeng	Rection Vol (mL)
500 -1,000 ng	Ho fihlela ho 12 ml

Tokisetso

1. Lokisetsa lisebelisoa tse latelang:
   

   Ntho	Polokelo	Taelo
   SureSelect XT HS le XT Low Input Blocker Mix	-25°C ho ea ho -15°C	Qhoma ka leqhoa. Vortex ho kopanya, ebe centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa.
   SureSelect RNase Block	-25°C ho ea ho -15°C	Qhoma ka leqhoa. Vortex ho kopanya, ebe centrifuge  ka bokhutšoanyane. Tsoela pele holim'a leqhoa.
   SureSelect Fast Hybridization Buffer	-25°C ho ea ho -15°C	Qhibiliha 'me u boloke mocheso oa kamore
   BOLELANG-Seq™ TargetSeq Blocker	-25°C ho ea ho -15°C	E qhibiliha ka mocheso oa kamore. Vortex ho kopanya, ebe centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa.
   BOLELANG-Seq™ DNA Library	-25°C ho ea ho -15°C	Qhibiliha 'me u lule leqhoeng

   SSel XT HS le XT Low Input Human All Exon V8

   -85°C ho ea ho -75°C

   Qhoma ka leqhoa. Vortex ho kopanya, ebe centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa.

2. Theha Lenane la Hybridization (HP) holim'a sebaesekele sa thermo (e nang le sekwahelo se futhumetseng ON) ka lenaneo le ka tlase. Qala lenaneo, 'me hang-hang u tobetse konopo ea Emisa, u lumelle sekoaelo se futhumetseng hore se fihlele mocheso ha u ntse u hlophisa maikutlo.

Nomoro ea Karolo	Palo ea Lipotoloho	Mocheso	Nako
1	1	95°C	metsotso e 5
2	1	65°C	metsotso e 10
3	1	65°C	motsotso o le 1
4	60	65°C 37°C	motsotso o le 1 3 metsotsoana
5	1	65°C	Tšoara

Tsamaiso

1. Beha 500–1000 ng ea laebrari e 'ngoe le e 'ngoe e lokisitsoeng ea TELL-Seq™ampkenya liliba tsa "strip tube" ebe u tlisa molumo oa ho qetela selibeng ka seng ho 12 µl u sebelisa metsi a se nang nuclease ha ho hlokahala. Hantle-ntle etsa kakaretso ampDNA ea laeborari ea TELL-Seq™, e bohareng ba 500-1000 ng 'me e sebelise tsohle bakeng sa karabelo ea hybridization.
2. Ho laeborari e 'ngoe le e 'ngoe ea TELL-Seq™ sample hantle, eketsa 5 µl ea SureSelect XT HS le XT Low Input Blocker Mix 'me u kenye 5 µl ea TELL-Seq™ Target Blocker. Koahela liliba ebe o vortex ka lebelo le phahameng bakeng sa metsotsoana e 5. Hlakola tube e hlobolang ka bokhuts'oane ho bokella mokelikeli o ntša lipululana.
3. Fetisetsa li-tubes ho baesekele e futhumatsang 'me u tobetse konopo ea Play ho qala lenaneo la HP hape.
   HLOKOMELA:
   Thermal cycler e tlameha ho emisoa nakong ea Karolo ea 3 (bona HP) ho lumella li-reagents tse eketsehileng hore li ekeletsoe liliba tsa Hybridization, joalokaha ho hlalositsoe mohatong oa 6. Nakong ea Karolo ea 1 le ea 2 ea lenaneo la libaesekele tse futhumetseng, qala ho lokisetsa li-reagents tse eketsehileng joalokaha ho hlalositsoe ho mohato oa 4 le oa 5. Haeba ho hlokahala, o ka qeta mehato ena ea boitokiso ka mor'a ho emisa sebaesekele sa mocheso Karolong ea 3.
4. Lokisetsa tharollo ea 25% ea SureSelect RNase Block (e nang le 1 volume ea RNase Block e nang le metsi a 3), ho latela tafole e ka tlase. Lokisetsa chelete e hlokahalang bakeng sa palo ea mekhoa e nyalisitsoeng ha nako e ntse e ea, hammoho le ho feta.
   

   Reagent

   Bolumo ho latela karabelo (mL)

   Bolumo bakeng sa 1 Reaction		Bolumo bakeng sa liketso tse 8 (ho kenyeletsoa ho feteletseng)	Bolumo bakeng sa liketso tse 24 (ho kenyeletsoa ho feteletseng)
   SureSelect RNase Block	0.5 mL	4.5 mL	12.5 mL
   Metsi a se nang metsi	1.5 mL	13.5 mL	37.5 mL

5. Lokisetsa Capture Library Hybridization Mix ka tatellano e latelang.
   

   Reagent	Bolumo ho latela karabelo (mL)
   	Bolumo bakeng sa 1 Reaction	Bolumo bakeng sa liketso tse 8 (ho kenyeletsoa ho feteletseng)	Bolumo bakeng sa liketso tse 24 (ho kenyeletsoa ho feteletseng)
   25% tharollo ea RNase Block	2 mL	18 mL	50 mL
   SSel XT HS le XT Low Input Batho bohle Exon V7	5 mL	45 mL	125 mL
   SureSelect Fast Hybridization Buffer	6 mL	54 mL	150 mL

   Kopanya li-reagents tse thathamisitsoeng ka mocheso oa kamore. Kopanya hantle ka ho vortexing ka lebelo le phahameng bakeng sa metsotsoana e 5 ebe o bilika hanyane. Tsoela pele hanghang ho mohato oa 6.

6. Hang ha sebaesekele se futhumatsang se qala Karolo ea 3 ea HP (motsotso o le mong ho 1°C), tobetsa konopo ea Khutsa. Ha baesekele e emisitsoe, 'me ha e ntse e boloka DNA + Blocker sampka har'a baesekele, fetisetsa 13 µl ea mocheso oa kamore Capture Library Hybridization Mix ho tloha mohato oa 6 ho isa ho o mong le o mongample hantle. Kopanya hantle ka ho kenya liphaephe holimo le tlase butle-butle ka makhetlo a 8 ho isa ho a 10. Liliba tsa hybridization hona joale li na le hoo e ka bang 35 μl
7. Etsa bonnete ba hore liliba tsohle li tiisitsoe ka ho feletseng. Vortex ka bokhutšoane, ebe o spin strip tube ka bokhuts'oane ho tlosa bubble leha e le efe ho tloha botebong ba liliba. Hang-hang khutlisetsa tube e hlobolang ho sebaesekele sa mocheso.
8. Tobetsa konopo ea Play ho qala hape lenaneo la libaesekele tse futhumetseng ho lumella hybridization ea DNA e lokisitsoeng.amples ho laebrari ea Capture.

Lokisetsa Streptavidin-coated Magnetic Beads

Lintho tse ka sebelisoang

• Dynabeads MyOne Streptavidin T1 (Mosebelisi)
• SureSelect Binding Buffer (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 1 (Post PCR), CAP)

Tokisetso

Lokisetsa lisebelisoa tse latelang:

Ntho	Polokelo	Taelo
Dynabeads MyOne Streptavidin T1	2°C ho isa ho 8°C	Centrifuge ka matla. Boloka mocheso oa kamore.
SureSelect Binding Buffer,	Mocheso oa Kamore	Boloka mocheso oa kamore.

Tsamaiso

1. Emisa hape ka matla lifaha tsa makenete tsa Dynabeads MyOne Streptavidin T1 holim'a motsoako oa vortex. Lifaha tsa makenete li lula nakong ea polokelo.
2. Bakeng sa hybridization e 'ngoe le e' ngoe sample, eketsa 50 µl ea lifaha tse tsosolositsoeng lilibeng tsa poleiti e ncha ea PCR kapa tube e hlobolang.
3. Hlatsoa lifaha ka ho eketsa 200 µl tsa SureSelect Binding Buffer. Kopanya ka pipetting holimo le tlase makhetlo a 20 kapa koala liliba le vortex ka lebelo le phahameng bakeng sa metsotsoana e 5-10.
4. Kenya poleiti kapa tšupu e hlobolang ka har'a sesebelisoa sa karohano sa makenete.
5. Ema bonyane metsotso e 5 kapa ho fihlela tharollo e hlakile, ebe o tlosa le ho lahla supernatant.
6. Pheta Mehato 3-5 ka makhetlo a mabeli bakeng sa ho hlatsoa ha 3 kaofela.
7. Emisa lifaha hape ka 200 µl tsa SureSelect Binding Buffer.

Tšoara DNA ea Hybridized u sebelisa Streptavidin-coated Bead

Lintho tse ka sebelisoang

• SureSelect Wash Buffer 1, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 1 (Post PCR), )
• SureSelect Hlatsoa Buffer 2, 16 Rxn (Agilent, SureSelect XT HS Target Enrichment Kit ILM Hyb Module, Box 1 (Post PCR), )

Tokisetso

Lokisetsa lisebelisoa tse latelang:

Ntho	Polokelo	Taelo
SureSelect Wash Buffer 1,	Mocheso oa Kamore	Boloka mocheso oa kamore.
SureSelect Wash Buffer 2,	Mocheso oa Kamore	Chesa li-aliquots tse 200 µl ho 70°C. Sheba Mohato oa 4

Tsamaiso

1. Ka mor'a hore mohato oa hybridization o phethoe 'me sebaesekele se chesang se fihle ho 65 ° C, fetisetsa s.ampka mocheso oa kamore.
2. Hang-hang fetisetsa molumo oohle (hoo e ka bang 30 µl) oa motsoako o mong le o mong oa hybridization ho liliba tse nang le 200 µl ea lifaha tsa streptavidin tse hlatsoitsoeng ka pipette e mengata. Pipette holimo le tlase makhetlo a 5-8 ho kopanya.
3. Eketsa tube ea strip ea ho hapa holim'a motsoako oa poleiti ea seliba sa 96, o kopanya ka matla (ka 1400-1800 rpm) kapa rotator, metsotso e 30 mocheso oa kamore. Etsa bonnete ba hore sampli-les li tsoakana hantle lilibeng.
4. Tsoela pele mohatong o latelang hang ka mor'a ho qhoqhoa. Nakong ea metsotso e 30 ea ho incubation bakeng sa ho tšoaroa, prewarm SureSelect Wash Buffer 2 at 70°C joalokaha ho hlalositsoe ka tlase. Beha li-aliquots tse 200 µl tsa Wash Buffer 2 lilibeng tsa poleiti e ncha ea 96- seliba kapa li-tubes tse hlobolang. Aliquot liliba tse 6 tsa buffer bakeng sa DNA ka 'ngoeample mathang. Koahela liliba ebe u e kenya ka har'a sebaesekele se futhumatsang, ka sekwahelo se futhumetseng, se ts'oaroe ka 70 ° C ho fihlela se sebelisoa.
5. Ha nako ea metsotso e 30 ea incubation e qalileng Mohato oa 3 e phethiloe, spin sampka bokhuts'oane ho bokella mokelikeli.
6. Beha tube e hlobolang ka har'a karohano ea makenete ho bokella lifaha. Emela ho fihlela tharollo e hlakile, ebe o tlosa le ho lahla supernatant.
7. Emisa lifaha hape ka 200 µl ea SureSelect Wash Buffer 1. Kopanya ka ho kenya lipeipi holimo le tlase makhetlo a 15-20, ho fihlela lifaha li emisitsoe ka botlalo.
8. Beha tube e hlobolang ka har'a karohano ea makenete. Emela hore tharollo e hlake (hoo e ka bang motsotso o le mong), ebe o tlosa le ho lahla supernatant.
9. Tlosa li-tubes tse hlobolang ho tloha karohanong ea makenete 'me u li fetisetse rack ka mocheso oa kamore. Emisa lifaha hape ka 200 µl ea 70°C e futhumetseng pele ho Wash Buffer 2. Pipette holimo le tlase makhetlo a 15-20, ho fihlela lifaha li emisitsoe ka botlalo. Koahela liliba ka li-caps tse ncha ebe u vortex ka lebelo le phahameng bakeng sa metsotsoana e 8. Hlakola poleiti kapa tube e hlobolang hakhutšoanyane ho bokella mokelikeli ntle le ho penta lifaha.
10. Emela sampfokotsa metsotso e 5 ho 70 ° C holim'a sebaesekele se chesang se nang le sekoahelo se futhumetseng.
11. Beha tube e hlobolang ka har'a karohano ea makenete ka mocheso oa kamore. Emela motsotso o le mong hore tharollo e hlake, ebe o tlosa le ho lahla supernatant.
12. Pheta Mehato ea 9-11 ka makhetlo a mang a mahlano, bakeng sa ho hlatsoa ha 6 kaofela.
13. Ka mor'a ho netefatsa hore lisebelisoa tsohle tsa ho hlatsoa li tlositsoe, eketsa 25 µl ea metsi a se nang nuclease sebakeng se seng le se seng.ample hantle. Pipette holimo le tlase makhetlo a 8 ho emisa lifaha hape. Samples e ka bolokoa holim'a leqhoa pele amptokafatso

Amplify Laebrari e hapiloeng

Lintho tse ka sebelisoang

• 5× Herculase II Reaction Buffer (Agilent, SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR), CAP Clear)
• Herculase II Fusion DNA Polymerase (Agilent, SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR), CAP Red)
• 100 mM dNTP Mix, (Agilent, SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR), CAP Green)
• SureSelect Post-Capture Primer Mix (Agilent, SureSelect XT HS Target Enrichment Kit, ILM Hyb Module Box 2 (Post PCR), CAP Clear)

Tokisetso

1. Lokisetsa lisebelisoa tse latelang:
   

   Ntho	Polokelo	Taelo
   5 × Herculase II Reaction Buffer	-25°C ho ea ho -15°C	Qhibiliha mocheso oa kamore. Vortex ho kopanya, ebe centrifuge hakhutšoanyane. Tsoela pele holim'a leqhoa.
   Herculase II Fusion DNA Polymerase	-25°C ho ea ho -15°C	Centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa.
   100 mM dNTP Motsoako	-25°C ho ea ho -15°C	Qhibiliha mocheso oa kamore. Vortex ho  kopanya, ebe centrifuge hakhutšoanyane. Tsoela pele holim'a leqhoa.
   SureSelect Post-Capture Primer Mix	-25°C ho ea ho -15°C	E qhibiliha ka mocheso oa kamore. Vortex ho kopanya, ebe centrifuge ka bokhutšoanyane. Tsoela pele holim'a leqhoa.

2. Beha lenaneo le latelang ho sebaesekele sa thermo ka tsela e latelang:
   • 98°C metsotso e 2
   • [98°C metsotsoana e 30, 63°C metsotsoana e 30, 72°C motsotso o le mong] x 1
   • 72°C metsotso e 5
   • 4°C ka ho sa feleng

Tsamaiso

1. Lokisetsa molumo o nepahetseng oa motsoako oa karabelo ea PCR. Kenya li-reagents tse latelang ho tšupu ea PCR ho latela palo ea maikutlo.
   

   Reagent	Bolumo ho latela karabelo (µL)
   	Bolumo bakeng sa 1 Reaction	Bolumo bakeng sa liketso tse 8 (ho kenyeletsoa ho feteletseng)	Bolumo bakeng sa liketso tse 24 (ho kenyeletsoa ho feteletseng)
   Metsi a se nang metsi	12.5µL	112.5µL	312.5µL
   5 × Herculase II Reaction Buffer	10µL	90µL	250µL
   Herculase II Fusion DNA Polymerase	1µL	9µL	25µL
   100 mM dNTP Motsoako	0.5µL	4.5µL	12.5µL
   SureSelect Post-Capture Primer Mix	1µL	9µL	25µL

2. Lokisetsa molumo o nepahetseng oa motsoako oa karabelo ea PCR. Kenya li-reagents tse latelang ho tšupu ea PCR ho latela palo ea maikutlo. Kenya 25 µl ea motsoako oa karabelo ea PCR o lokiselitsoeng ho Lethathamo la 29 ho lets'oao ka lengample seliba se nang le 25 µl ea DNA e ruisitsoeng ka lifaha (e lokisitsoeng leqepheng la 26 'me e tšoaretsoe holim'a leqhoa).
3. Kopanya karabelo ea PCR hantle ka ho penya holimo le tlase ho fihlela ho emisoa ha lifaha ho tšoana. Qoba ho fafatsa samples holim'a mabota a seliba; u se ke ua ohla samphanyane ka mohato ona.
4. Beha tube holim'a sebaesekele sa mocheso 'me u tsamaise lenaneo (sheba ka holimo) ka palo e nepahetseng ea lipotoloho.
5. Ha PCR e amplenaneo la lification le phethehile, spin tube ea hlobolisa ka bokhutšoanyane. Tlosa lifaha tse koahetsoeng ke streptavidin ka ho beha poleiti kapa tube e hlobolang holim'a sethala sa makenete mocheso oa kamore. Emela metsotso e 2 hore tharollo e hlake, ebe u tlosa ntho e 'ngoe le e 'ngoe e phahametseng tlhaho (e ka bang 50 µl) ho ea lilibeng tsa tube e hlobolang.

Hloekisa Laeborari e Hapiloeng 

Lintho tse ka sebelisoang

• AMPure XP (Mosebelisi)
• Bopaki ba Ethanol 200 (bo felletseng) bakeng sa baeloji ea limolek'hule (Mosebelisi)
• Metsi a se nang nyutlelie (Mosebelisi)
• TE buffer, pH 8.0 (Mosebelisi)
• 0.2 mL PCR tube kapa strip tube (Mosebelisi)

Tokisetso

Lokisetsa lisebelisoa tse latelang:

Ntho	Polokelo	Taelo
Fresh 75% (v/v) ethanol	Mocheso oa Kamore	E hloka 400 ml ka motsotsoanaample. Kopanya 1.5 mL Ethanol (bopaki ba 200) le 0.5 mL metsi a se nang Nuclease. Vortex ho kopanya le ho boloka mocheso oa kamore.
AMPke XP	2°C ho isa ho 8°C	E tlise mocheso oa kamore bonyane metsotso e 20 le vortex ka matla ho emisa lifaha pele li sebelisoa.
Metsi a se nang metsi	Mocheso oa Kamore	Boloka mocheso oa kamore.
TE buffer, pH 8.0	Mocheso oa Kamore	Boloka mocheso oa kamore.

Tsamaiso

1. Theola tharollo ka lebelo la ~ 1 motsotsoana o potoloha ka har'a centrifuge.
2. Vortex ka matla ho emisa hape ho AMPure XP mme o kenye 50 μL AMPKenya XP sehlahisoa se seng le se seng sa PCR.
3. Kopanya ka liphaephe holimo le tlase makhetlo a 10.
4. E kenye ka mocheso oa kamore ka metsotso e 5.
5. Beha tube holim'a sethala sa makenete bakeng sa motsotso o le 1 kapa ho fihlela tharollo e hlakile.
6. Aspirate le lahla supernatant ntle le ho khathatsa AMPure lifaha.
7. Ha u ntse u boloka pompo e eme sebakeng sa makenete, eketsa 200 µL e sa tsoa lokisoa e 75% ea ethanol ka har'a tube. E lumelle ho lula metsotsoana e 30.
8. Aspirate le lahla matla a phahametseng tlhaho ntle le lifaha tse sitisang.
9. Pheta mehato ea 7-8 nako e 'ngoe hape, u boloke tube e eme ka matla a khoheli nako eohle.
10. Tlohela tšupu holim'a sethala sa makenete se butse kepisi 'me u lumelle tšupu hore e ome ka metsotso e 1-2 ho etsa mouoane mesaletsa ea ethanol. U SE KE UA omisa lifaha ho feta tekano.
11. Tlosa tšupu sebakeng sa makenete ebe u eketsa 25 µL TE buffer ho lifaha.
12. Pipette kapa vortex ho emisa lifaha hape. E lumelle ho lula metsotso e 5.
13. Beha tube holim'a sethala sa makenete bakeng sa motsotso o le 1 kapa ho fihlela tharollo e hlakile.
14. Fumana 23 µL ea matla a holimo ho tube e ncha. Hlokomela hore u se ke ua senya lifaha.
15. Supernatant e na le laeborari e hapiloeng ea TELL-Seq™.

HLOKOMELA:
Ena ke TLHOKOMELISO EA MAEMO. Laeborari e hapiloeng ea TELL-Seq e ka bolokoa ho -25°C ho ea ho -15°C ka likhoeli tse tšeletseng.

Tšoaneleha 'me U Lekanye Laebrari e Hapiloeng bakeng sa Tatellano

Lintho tse ka sebelisoang

• Agilent Bioanalyzer High Sensitivity DNA Kit kapa TapeStation High Sensitivity D5000 ScreenTape Assay (Mosebelisi)
• Qubit dsDNA HS Assay Kit (Mosebelisi)
• TE buffer, pH 8.0 (Mosebelisi)

Tokisetso

1. Lokisetsa lisebelisoa tse hlokahalang ho latela Bioanalyzer kapa TapeStation le Qubit.

Tsamaiso

1. Sebelisa 1 µL ea laebrari bakeng sa Agilent Bioanalyzer High Sensitivity DNA Kit kapa 2 µL ea laeborari bakeng sa TapeStation High Sensitivity D5000 ScreenTape Assay.
2. Ho tseba hore na laeborari e hokae, beha Setereke ho software ea tlhahlobo ea Bioanalyzer kapa TapeStation ho tloha ho 150 bp ho isa ho 1000 bp. Rekota sample Concentration (nM) sebakeng sena (sheba setšoantšo sa 2). Ho tseba boholo ba laeborari, beha sebaka ho tloha ho 150 bp ho isa ho 3000 bp. Rekota sample Karolelano ea Boholo (bp) joalo ka Boholo ba Laebrari.
   TLHOKOMELO
   'Malo oa mahloriso o tsoang ho Bioanalyzer (kapa TapeStation) o lokela ho sebelisoa e le qalo ea ho etsa dilution kapa pokello ea laeborari bakeng sa tatellano. Netefatsa hore na laeborari ea ho qetela e hlapolotsoeng ea tatellano kapa letamo la pokello ea libuka ka Qubit dsDNA HS Assay kit (sheba Mohato oa 6).Setšoantšo sa 2. Example of exume captured library profile ho tsoa tekong ea Tape Station High Sensitivity D5000 Screen Tape.
3. Laeborari e ka hlophiswa hanghang kapa ya bolokwa ho -25°C ho ya ho -15°C.
4. Ha o hlophisa, hlapolla laeborari o sebelisa TE buffer ho ea mahloriso a khothaletsoang ke sistimi e 'ngoe le e' ngoe ea tatellano ea Illumina®. Etsa pokello e hlapolotsoeng ea laeborari bakeng sa tatellano haeba libuka tse fetang bonngoe li tla hlophisoa ka nako e tšoanang.
5. Lekanya pokello ea libuka ka Qubit dsDNA HS Assay Kit. Sebelisa boleng ba Average Size ho tsoa ho tekanyo ea Bioanalyzer (kapa TapeStation) joalo ka boholo ba laeborari bakeng sa ho fetolela boima ba 'mele ho ba molar concentration (nM).

A = Boima ba Boima (ng/µL)
S = Boholo ba Laeborari (bp)
Molar Concentration (nM) = (A*1,000,000)/(S*650)
Fetola molumo o hlokahalang ha ho hlophisoa tatellano haeba pokello ea laeborari e lekantsoeng ke Qubit e fapane le e khothaletsoang ho feta 10%.

Litokomane / Lisebelisoa

tatellano ea bokahohleng BOLELA-Seq Boikemisetso ba Boikemisetso [pdf] Bukana ea Mosebelisi Bolella-Seq Boikemisetso ba Maiketsetso, TELL-Seq, Boikemisetso ba Maikemisetso, Matlafatso

Litšupiso

• Bukana ea Mosebelisi