Revision as of 01:30, 12 July 2011 by >Sam Chiang
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
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
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
'
|
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
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
- 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
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
- 15 cycles
Size selection