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==Sequencing Library Construction using N2 adapter protocol== * Library ID: Various = DD-BSPP-DMR220K.N2-VarInd1to10-Sep23 (10 barcode, Note: Ind1-Ind4 prepared [http://genome-tech.ucsd.edu/LabNotes/index.php/Dinh/NOTES/2010-8-22] [John Hopskins, 4 samples] * Library ID: Sergio = DD-BSPP-DMR220K.N2-SergioInd1to12-Sep23 (12 barcode) {| {{table}} border=1 | align="center" style="background:#f0f0f0;"|'''ID''' | align="center" style="background:#f0f0f0;"|'''Sample Name''' | align="center" style="background:#f0f0f0;"|'''Conc(ng/ul)''' | align="center" style="background:#f0f0f0;"|'''MmeI Vol(ul) DNA''' | align="center" style="background:#f0f0f0;"|'''MmeI Vol(ul) H2O''' | align="center" style="background:#f0f0f0;"|'''Barcode used''' | align="center" style="background:#f0f0f0;"|'''Conc(ng/ul) after library amp''' | align="center" style="background:#f0f0f0;"|'''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| 400px]] [[File:DD 2010-09-07 Capture Amplicons Baylor, Sergio, JH, PGPfib B.jpg | 400px]] * 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.jpg |400px]][[File:DD_2010-09-07_MmeI_10-17.jpg |400px]] [[File:DD_2010-09-07_MmeI_18-21.jpg |400px]] 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.jpg|400px]][[File:ZhangLab 2 2010-09-08 MmeI repeat 10-18.jpg|400px]] 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 |400px]] '''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|200px]] '''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).
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