Revision as of 23:10, 22 January 2013 by >Zsakura2
WGBS of Xu mouse SCNT and iPSC samples
End repair
- Total Volume 70ul, sheared DNA 50ul, reaction mix 20ul
NEB End repair mix
|
1x rxn
|
13x rxn mix
|
10X End repai buffer |
7.00 |
91.00
|
NEB End repair enzyme |
5.00 |
65.00
|
H2O |
8.00 |
104.00
|
Total |
20.00 |
260.00
|
- Aliquot to column #1:
- A1-D1 ~ 42ul
- E1-H1 ~21ul
A-tailing
A-tailing reaction mix
|
1x rxn
|
13x rxn mix
|
NEB 10X A-tailing buffer |
3.00 |
39.00
|
A-tailing enzyme |
3.00 |
39.00
|
H2O |
24.00 |
312.00
|
Total |
30.00 |
390.00
|
- Aliquot to column #2:
- A2-D2 ~ 63ul
- E2-H2 ~31ul
Adapter ligation
- Total volume 50ul, A-tail reaction mix 40ul, methylation adapters 10ul
Ligation reaction mix
|
1x rxn
|
13x rxn mix
|
5x Quick Ligation Reaction buffer |
10.00 |
130.00
|
Quick T4 DNA ligase |
4.00 |
52.00
|
H2O |
26.00 |
338.00
|
Total |
40.00 |
520.00
|
- Aliquot to column #3:
- A3-D3 ~ 83 ul
- E3-H3 ~41ul
Methylation adapter preparation
- Use 10ul of methylated adapter to 5ug gDNA insert
- Use 3ul for 1.5ug DNA insert
- Note: We don't have enough adapter, so I reduced to 2ul
Prepare adapter |
2.0ulx 13rxn |
26.00
|
H2O |
7.0ulx13rxn |
91.00
|
Total |
|
117.00
|
- Aliquot to column #4:
- A4-D4 ~ 22 ul
- E4-H4 ~11ul
sample ID
|
N2 Index used)
|
2A4F1 |
N2 index 1
|
2A4F33 |
N2 index 2
|
miPS D1 |
N2 index 3 (not sequenced)
|
miPS B3 |
N2 index 4
|
1E12P20 |
N2 index 5
|
nt ES P7 C |
N2 index 6
|
nt ES P8 B |
N2 index 7
|
NB 1 |
N2 index 8 (not sequenced)
|
NB 3 |
N2 index 9
|
SCNT B12 |
N2 index 10
|
B6 ES |
N2 index 11
|
129 ES |
N2 index 12
|