Alice:Exome/2012-12-14
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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]
- Total volume 50ul
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