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==Prepare RNAseq library for sequencing== * Continue with lab note on 7/8/11 * Samples: PC1, PC2, GFP+, GFP-, E9.5, E11.5, E13.5m, E13.5f * 75ul sheared DNA store at 0.5ml non-stick tube over the weekend * Use NEB master mix kit for Library prep ===End repair=== '''Reaction''' sheared DNA 75ul Master mix buffer 10ul Enzyme mix 5ul H2O 10ul '''Total 100ul''' 20C for 30min '''Purification with AMPure beads''' # 150ul beads to 100ul reaction (kapa lib prep kit suggested), mix 10 times, R.T for 8min # Place on magnetic stand for 5min # Remove sup., wash with 180ul fresh made 80% EtOH twice # Air-dry for 15min, re-suspend with 32ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min # Transfer 30ul DNA from beads {| {{table}} | align="center" style="background:#f0f0f0;"|'''After end''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''ng''' |- | GFP+||0.35||10.5 |- | GFP-||0.24||7.2 |- | E9.5||0.39||11.7 |- | E11.5||1.29||38.7 |- | E13.5m||0.54||16.2 |- | E13.5f||2.27||68.1 |- | |} ===A tailing=== '''Reaction''' End-repaired DNA 30ul Master mix buffer 5ul Enzyme mix 3ul H2O 12ul '''Total 50ul''' 37C for 30min '''Purification with AMPure beads''' # 90ul beads to 50ul reaction, mix 10 times, R.T for 8min # Place on magnetic stand for 5min # Remove sup., wash with 180ul fresh made 80% EtOH twice # Air-dry for 15min, re-suspend with 20ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min # Transfer 18ul DNA from beads, 1ul left in tube for Qubit measurement ===Adapter ligation=== * 30uM Y-adapter made by Alice few weeks ago * 0.08ul for sample 1,3,4,5,7 and 0.4ul for 2,6,8 * 50ul total with 10ul buffer and 5ul Quick T4 ligase; RT for 30min {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''ng''' | align="center" style="background:#f0f0f0;"|'''ug''' | align="center" style="background:#f0f0f0;"|'''pmole''' | align="center" style="background:#f0f0f0;"|'''adaptor''' | align="center" style="background:#f0f0f0;"|'''ul (30uM)''' |- | PC2||0.91||16.4||0.016||0.12||2.42||0.08 |- | GFP+||0.29||5.27||0.005||0.04||0.76||0.03 |- | GFP-||0.23||4.14||0.004||0.03||0.6||0.02 |- | E9.5||0.4||7.22||0.007||0.05||1.06||0.04 |- | E11.5||1.61||28.98||0.029||0.22||4.4||0.15 |- | E13.5m||0.48||8.59||0.009||0.07||1.36||0.05 |- | E13.5f||2.72||48.96||0.049||0.37||7.42||0.25 |- | |} '''Purification with AMPure beads''' # 50ul beads to 50ul reaction, mix 10 times, R.T for 8min # Place on magnetic stand for 5min # Remove sup., wash with 180ul fresh made 80% EtOH twice # Air-dry for 15min, re-suspend with 22ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min # Transfer 20ul DNA from beads, 1ul left in tube for Qubit measurement ===PCR amplification=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''ng''' |- | PC1||0.15||2.7 |- | PC2||0.87||15.61 |- | GFP+||0.25||4.52 |- | GFP-||0.24||4.3 |- | E9.5||0.31||5.51 |- | E11.5||1.22||21.96 |- | E13.5m||0.51||9.22 |- | E13.5f||2.63||47.34 |- | |} ====Pre-run==== * Pre-run with PC1 (lowest amount of template) and PC2 (relatively high amount of template) * 50ul total: 25ul Phusion master mix, 1ul 10uM PCR primer each * 98C 30sec; 98C 10sec, 60C 30sec, 72C 30sec (15 cycles); 72C 5min [[File:7.11.11_pre-run.jpg]] [[File:7.11.11_pre-run gel check.jpg]] Something wrong with the loading buffer, use a new one! ====Sample run==== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''ng/ul''' | align="center" style="background:#f0f0f0;"|'''ul''' | align="center" style="background:#f0f0f0;"|'''ng''' |- | PC1||0.15||18||2.7 |- | PC2||0.87||18||15.61 |- | GFP+||0.25||10||2.5 |- | GFP-||0.24||10||2.4 |- | E9.5||0.31||10||3.1 |- | E11.5||1.22||2||2.4 |- | E13.5m||0.51||5||2.5 |- | E13.5f||2.63||1||2.6 |- | |} * 50ul total: 25ul Phusion master mix; 1ul 10uM Pf; 1ul 10uM Pr.ID1 to 6 * 98C 30sec; 98C 10sec, 60C 30sec, 72C 30sec (12 cycles); 72C 5min QPCR curve [[File:7.11.11_qpcr.jpg]] ===Size selection=== Continue with lab note on 7.12.11 [http://genome-tech.ucsd.edu/LabNotes/index.php/Rui:LabNotes/Collaborations/2011-7-12]
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