Revision as of 01:40, 7 March 2014 by >Zsakura2
WGBS of Xu mouse SCNT and iPSC samples[edit]
- sheared 3ug of gDNA for the samples below, and used 1.5 ug for WGBS sample prep
sample ID
|
N2 Index used
|
cell type
|
progenitor line
|
progenitor/donor mouse strain
|
2A4F1 |
N2 index 1 |
iPSC |
B6MEF (we don't have the original sample) |
C57BL/6J
|
2A4F33 |
N2 index 2 |
iPSC |
B6MEF (we don't have the original sample) |
C57BL/6J
|
miPS D1 |
N2 index 3 (not sequenced) |
iPSC |
ZN_B6MEF |
C57BL/6J
|
miPS B3 |
N2 index 4 |
iPSC |
ZN_B6MEF |
C57BL/6J
|
1E12P20 |
N2 index 5 |
iPSC |
TB_B6MEF |
C57BL/6J
|
nt ES P7 C |
N2 index 6 |
SCNT |
B6MEF P0-3 |
C57BL/6J
|
nt ES P8 B |
N2 index 7 |
SCNT |
B6MEF P0-3 |
C57BL/6J
|
NB 1 |
N2 index 8 (not sequenced) |
SCNT |
B6-P1-1 |
C57BL/6J
|
NB 3 |
N2 index 9 |
SCNT |
B6-P1-1 |
C57BL/6J
|
SCNT B12 |
N2 index 10 |
SCNT |
B6-P1-2 |
C57BL/6J
|
B6 ES |
N2 index 11 |
ES |
- |
C57BL/6J
|
129 ES |
N2 index 12 |
ES |
- |
-
|
End repair[edit]
- 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[edit]
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[edit]
- 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[edit]
- 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 |
3.0ulx 13rxn |
39.00
|
H2O |
7.0ulx13rxn |
91.00
|
Total |
|
130.00
|
- Aliquot to column #4:
- A4-D4 ~ 22 ul
- E4-H4 ~11ul
- Add 40ul of Ligation reaction mix
- Mix by pipetting 10 times
- Add 10ul of diluted Methylation adapters
- Mix by pipetting 10 times
- Shake at 1000 rpm for 5min (lid on)
- Incubate at 20C for 15min
- Add 50ul of 20%PEG/2.5M NaCl
- Mix at 750rpm for 5min, 25C (lid on)
- Incubate at 25C for 5 min
- Put on magnet and discard spnt
- Wash twice with 140ul 80% EtOH
- Dry the beads
- Resuspend adapter ligated DNA with ~23ul EB buffer
Bisulfite conversion using EZ-96 DNA Methylation-Lightning™ MagPrep[edit]
Performed bisulfite conversion manually
Elute with 40ul Elution buffer (final volume ~35ul)
Amplification of bisulfite sequencing libraries with N2 barcoded primers[edit]
Perform in 50ul reaction with PCR_R_Ind 1-12, add 7ul of adapter-ligated bis-cvt DNA
Reagnets 1rxn 13 rxn mix
Adapter ligated bis-cvt DNA 10.00
10uM PCR_F 2.00 26
10uM PCR_R.Ind1-12 2.00
2x iQ MM 50.00 650
H2O 36.00 468
Total 100.00 1300
98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 13 cycles --> 72C 3min --> 15C Hold
Qiaquick column purification and elute in 42ul H2O
PAGE gel quantification and size selection[edit]
sample ID
|
N2 Index used
|
amount needed for pooling (ul)
|
2A4F1 |
N2 index 1 |
5.08
|
2A4F33 |
N2 index 2 |
7.05
|
miPS D1 |
N2 index 3 (not sequenced) |
-
|
miPS B3 |
N2 index 4 |
7.77
|
1E12P20 |
N2 index 5 |
7.43
|
nt ES P7 C |
N2 index 6 |
12.5
|
nt ES P8 B |
N2 index 7 |
33.17
|
NB 1 |
N2 index 8 (not sequenced) |
-
|
NB 3 |
N2 index 9 |
4.95
|
SCNT B12 |
N2 index 10 |
4.88
|
B6 ES |
N2 index 11 |
4.88
|
129 ES |
N2 index 12 |
6.75
|
Sequencing and read mapping summary[edit]
- The samples were sequenced in Hiseq 130104_SN1001
- The mapping included reads from Hiseq 130104_SN1001 and also lane 1 from the GA run 121226_HL140
Sample IDs
|
Experiment
|
Index number
|
Total reads
|
Mapped reads
|
Mapping rate
|
Conversion rate*
|
Ave. coverage
|
2A4F1 |
WGBS |
Indx01 |
453017 |
328106 |
0.724268626 |
0.98 |
10.6
|
2A4F33 |
WGBS |
Indx02 |
409103 |
293128 |
0.716513934 |
0.984 |
9.4
|
miPS B3 |
WGBS |
Indx04 |
406101 |
278669 |
0.686206141 |
0.989 |
7.8
|
1E12P20 |
WGBS |
Indx05 |
375114 |
256596 |
0.684048049 |
0.988 |
8.4
|
nt ES P7 C |
WGBS |
Indx06 |
359991 |
250016 |
0.694506252 |
0.99 |
7.6
|
nt ES P8 B |
WGBS |
Indx07 |
517166 |
362510 |
0.700954819 |
0.991 |
12.3
|
NB 3 |
WGBS |
Indx09 |
397456 |
279016 |
0.70200475 |
0.986 |
7.9
|
SCNT B12 |
WGBS |
Indx10 |
362114 |
250918 |
0.692925432 |
0.982 |
8.5
|
B6 ES |
WGBS |
Indx11 |
352934 |
245005 |
0.694194949 |
0.986 |
7.9
|
129 ES |
WGBS |
Indx12 |
322470 |
200374 |
0.621372531 |
0.989 |
5.7
|
|
File:Hclusts-10kb-ZL-mSCNT-WGBS.png