Alice:LabNotes/2009-9-15
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Single-end solexa sequencing library[edit]
Materials[edit]
use previously captured samples capture reaction setup can be found under LabNotes 8-15-09 and 8-16-09 samples to be used: D3(6&7 subsets of NA12878), DF6-9-9, foreskin, ips(PGP1-ips), Cvi(CV-ips), CVf(CV-fibroblast) Check LabNotes 9-14-09 for the details on re-amplification PCR and the ratio to pool all the products together
Procedure[edit]
Reagent setup: (Pair-end sequencing library construction) Solexa_1 adaptor and Solexa_2_PE adaptor will be used for PCR at the last step use these primers: Solexa_PCR_PE_loH & Solexa_PCR_upH (single-end sequencing library construction) Solexa_1 adaptor and Solexa_2 adaptor will be used for PCR at the last step use these primers: Solexa_PCR_up Solexa_PCR_lo
PCR re-amplification[edit]
AmpF6.3/AmpR6.3 and dUTP:dNTP 1:40 system setup: x4 H2O 46ul 184ul 2x KAPA Master mix 50ul 200ul dUTP(1mM) 4ul 16ul AmpF6.3NH2(100uM) 0.4ul 8ul AmpR6.3NH2(100uM) 0.4ul 8ul template 0.5ul 2ul 95C 30sec -> (95C 3sec -> 60C 30sec) x 3 -> 60C 2min -> 15C hold.
Qiaquick column purification and elute in 30ul EB. Nanodrop: D3: 19.2ng/ul DF: 33.3ng/ul fs: 17.7ng/ul ips: 13ng/ul cvi: 23.7ng/ul cvf: 12.2ng/ul
Digestion with MmeI[edit]
(dUTP 250uM,167.3ng/ul of product will use 6ul of DNA along with 2ul of 1mM SAM and 8ul of 2U/ul MmeI) DF & cvi ips & cvf D3 & fs DNA 30ul 30ul 30ul 10X NEBuffer 4 5ul 4ul 5ul 1mM SAM(fresh) 6ul 3ul 4ul 2U/ul Mme I 8ul 3ul 5ul ddH2O 1ul - 6ul 1mM SAM: 32mM SAM 1ul + 31ul ddH2O. 37C 2h
MinElute column purify. Elute in 11ul EB.
USER digestion[edit]
DNA 10ul USER 3ul total 13ul 37C 4hr
S1 nuclease digestion[edit]
10 x S1 nuclease buffer: 2ul DNA after USER digestion: 13ul S1 nuclease (10U/ul): 1ul ddH2O 4ul
37C 10mins. Minelute cloumn purify. Elute in 16ul H2O.
end repair[edit]
Total 25ul DNA 15ul dNTP 5ul 10xendrepair buffer 2.5ul enzyme 1ul ATP(10mM) 5ul Keep at room temperature (~25C) for 45 minutes. Purify with Minelute. Elute in 20ul H2O. PAGE size selection File:ZhangLab 2 2009-09-16 12hr 47min.jpg File:ZhangLab 2 2009-09-16 12hr 48min.jpg
adapter ligation[edit]
adaptor construction: add 10ul Solexa_2_PE_up and 10ul Solexa_2_PE_lo_noP. 10ul Solexa_2_upNH2 and 10ul Solexa_2_lo_noP. 10ul Solexa_1_upNH2 and 10ul Solexa_1_lo_noP. 95C 5mins -> 65C 30mins -> 4C.
total 30ul DNA 13ul 100uM Solexa_1 adaptor 1ul 100uM Solexa_2 adaptor 1ul 2xQuickLiage buffer 15ul QuickLigase enzyme(NEB) 1ul extra ATP(10mM) 2.5ul Keep at room temperature (~25C) for 10 minutes. Purify with Minelute column. Elute in 10ul H2O.
Nick-translation[edit]
DNA 10ul 10x ThermoPol buffer 1.2ul 10mM dNTP 0.5ul Bst polymerase(8U/ul) 0.5ul
65C for 12 minutes
PCR of sequencig library[edit]
Nick-translated DNA 6ul Solexa_PCR_up(100uM) 0.2ul Solexa_PCR_lo(100uM) 0.2ul 2xiProof master mix 50ul 50x SYBG 0.2ul ddH2O 44ul 98C 30sec -> 7-8 cycles of (98C 10sec -> 65C 20 sec -> 72C 20sec) -> 72C 3min ->15C hold. PAGE size selection 175-225bp File:ZhangLab 2 2009-09-17 16hr 21min-1.jpg
QPCR[edit]
template 1ul Phusion 2x master mix 25ul Syb FP5(100uM) 0.2ul Syb RP7(100uM) 0.2ul 50X SYBR green 0.2ul H2O 24ul
98C 30sec -> (98C 10sec -> 58C 20sec -> 72C 20 sec)X21 -> 72C 3min -> 15C
results: DF6-9-9: 12.3nM foreskin: 5nM CV-ips: 26.3nM CV-fibroblast: 10nM