Rui:LabNotes/Collaborations/2011-7-11

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Prepare RNAseq library for sequencing[edit]

  • 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[edit]

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
After end ng/ul 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[edit]

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[edit]

  • 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
' ng/ul ng ug pmole adaptor 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[edit]

Sample ng/ul 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[edit]

  • 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[edit]

Sample ng/ul ul 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[edit]

Continue with lab note on 7.12.11 [1]