Revision as of 21:50, 11 July 2011 by >RuiLiu
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
Sample
|
Concentration
|
ng/ul
|
GFP+ |
0.35 |
ug/mL
|
GFP- |
0.24 |
ug/mL
|
E9.5 |
0.39 |
ug/mL
|
E11.5 |
1.29 |
ug/mL
|
E13.5m |
0.54 |
ug/mL
|
E13.5f |
2.27 |
ug/mL
|
|
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 19ul Qiagen EB, mix 10 times, RT 2min, magnetic stand 2min
# Transfer 17ul DNA from beads, 1ul left in tube for Qubit measurement
Adapter ligation
'
|
ng/ul
|
ng
|
ug
|
pmole
|
adaptor
|
ul (30uM)
|
PC |
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
|
|
PCR amplification
Size selection