Dinh/NOTES/2010-9-6
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Sequencing Library Construction using N2 adapter protocol[edit]
- Library ID: Various = DD-BSPP-DMR220K.N2-VarInd1to10-Sep23
(10 barcode, Note: Ind1-Ind4 prepared [1] [John Hopskins, 4 samples]
- Library ID: Sergio = DD-BSPP-DMR220K.N2-SergioInd1to12-Sep23
(12 barcode)
ID | Sample Name | Conc(ng/ul) | MmeI Vol(ul) DNA | MmeI Vol(ul) H2O | Barcode used | Conc(ng/ul) after library amp | Library ID |
1 | hNSC | 11.5 | 21.7 | 25.8 | Ind1 | 24.0 | Sergio |
2 | hNiPS2Fp23 | 9.2 | 27.2 | 20.3 | Ind2 | 11.0 | Sergio |
3 | HFF-XF | 12.1 | 20.7 | 26.8 | Ind3 | 12.2 | Sergio |
4 | FiPS3F-1 | 7.6 | 32.9 | 14.6 | Ind4 | 13.2 | Sergio |
5 | HFIPS4F-7p9 | 7.8 | 32.1 | 15.4 | Ind5 | 15.8 | Sergio |
6 | KiPS4F-8 | 11.6 | 21.6 | 25.9 | Ind6 | 12.4 | Sergio |
7 | K-MMTA | 14.9 | 16.8 | 30.7 | Ind7 | 15.6 | Sergio |
8 | hKiPS3F-7p24 | 7.6 | 32.9 | 14.6 | Ind8 | 16.3 | Sergio |
9 | ASTiPS4F-5 | 15.8 | 15.8 | 31.7 | Ind9 | 19.5 | Sergio |
10 | Astrocytes | 13.6 | 18.4 | 29.1 | Ind10 | 13.4 | Sergio |
11 | ASTiPS4F-4 | 18.3 | 13.7 | 33.8 | Ind11 | 15.6 | Sergio |
12 | HUVECp2 | 14.2 | 17.6 | 29.9 | Ind12 | 22.6 | Sergio |
13 | HUViPS4F-1p20 | 15.1 | 16.6 | 30.9 | Ind5 | 12.8 | Various |
14 | HUViPS4F-3p | 15.2 | 16.4 | 31.1 | Ind6 | 16.5 | Various |
15 | L08-0020-T | 10 | 25.0 | 22.5 | None | ||
16 | L08-0016-T | 5.4 | 46.3 | 1.2 | None | ||
17 | L08-0050-T | 10.7 | 23.4 | 24.1 | None | ||
18 | 614T | 7.5 | 33.3 | 14.2 | Ind7 | 7.4 | Various |
19 | 614N | 6.9 | 36.2 | 11.3 | Ind8 | 8.3 | Various |
20 | PGP1fib | 9.7 | 25.8 | 21.7 | Ind9 | 7.9 | Various |
21 | PGP1iPS | 9.3 | 26.9 | 20.6 | Ind10 | 11.1 | Various |
22 | NTC | 0 | 0.0 | 47.5 | --- | ||
Aug22 | 499T | Ind1 | Various | ||||
Aug22 | 499N | Ind2 | Various | ||||
Aug22 | 536T | Ind3 | Various | ||||
Aug22 | 536N | Ind4 | Various |
File:DD 2010-09-07 Capture Amplicons Baylor, Sergio, JH, PGPfib.jpg File:DD 2010-09-07 Capture Amplicons Baylor, Sergio, JH, PGPfib B.jpg
- Used 250ng capture amplicon for library construction
MmeI Digestion
Reagent Volume(ul) DNA+H2O 47.5 MmeI 2 (4units) 10xNEB Buffer4 6 SAM (1mM) 4.5 -------------------------------------- Total 60 ul Loaded 1ul per sample File:DD 2010-09-07 MmeI 1-9.jpgFile:DD 2010-09-07 MmeI 10-17.jpg File:DD 2010-09-07 MmeI 18-21.jpg Purified using 0.9x AMPure beads. Elute using 40ul EB.
Repeat MmeI Digestion
Reagent Volume(ul) DNA 39 H2O 2.5 MmeI 1 (4units) 10xNEB Buffer4 5 SAM (1mM) 2.5 -------------------------------------- Total 50 ul Sample no. 1-18 Left to Right Loaded 1ul per sample File:DD 2010-09-08 MmeI repeat 1-9.jpgFile:ZhangLab 2 2010-09-08 MmeI repeat 10-18.jpg Purified using 0.9x AMPure beads. Elute using 40ul EB.
N2 Adapters Ligation
Reagent Volume(ul) DNA 39 QuickLigase 1 N2 adapters(0.5uM) 0.4 QuickLigase buffer 2x 40.4 ----------------------------------------- Total: 80.8 ul Purified using 0.7x Ampure Beads. Elute using 40ul EB.
RT_PCR Amplification using barcoded/non-barcoded Solexa amplification primers
- Proceeded using Baylor samples (no.15-17).
Reagent Volume(ul) DNA 40 PCR_F 4 PCR_R 4 SYBR 50x 0.8 H2O 52 2xPhusionHF 100 --------------------------------------- Total: 200.8ul = divide to 2 reactions per sample. Purify using Qiaquick 1 column per sample. Elute with 30ul EB.
Size selection using E-gel size-select 2%
- Loaded 25ul of each sample in each well.
File:DD 2010-09-09 Baylor seq lib e-gel ss.jpg
Reamplify using Syb_FP5 and Syb_RP7
Reagent Volume(ul) DNA 46ul Syb_FP5 2 Syb_RP7 2 2xPhusionHF 50ul ----------------------- Total: 100ul 98C (30s) -> (98C (3s)->62C (20s)->72C(20s)) x 4/5 -> 72C 2min->4C forever Purify using 0.9x AMPure beads. Elute with 40ul EB. Loaded 1ul per sample File:DD 2010-09-09 Baylor seq lib re-amp 1.jpg
Reamplify no.16 and 17 with RT_PCR
Reagent Volume(ul) DNA 40ul Syb_FP5 2 Syb_RP7 2 SYBR 50x 0.4x H2O 6 2xPhusionHF 50ul ----------------------- Total: 100ul 98C (30s) -> (98C (3s)->62C (20s)->72C(20s)) x 5 -> 72C 2min->15C forever Purify using 1 Qiaquick column each. Then re-purified using 0.8x AMPure beads. Elute with 40ul EB
Nanodrop: 15 (L08-0020T): 0.1ng/ul 16 (L08-0016T): 0.6ng/ul 17 (L08-0050T): 0.9ng/ul
PAGE Quant: no. ng/ul nM 15 0.38 1.86 16 0.83 4.06 17 0.99 4.85 Loaded 4ul per sample File:DD 2010-09-09 Baylor seq lib re-amp 2 quant.jpg
RT_PCR Amplification using barcoded/non-barcoded Solexa amplification primers
- Proceeded using Baylor samples (no.1-14 and 18-21).