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).