Alice:Exome/2012-12-14: Difference between revisions
Jump to navigation
Jump to search
>Zsakura2 (Created page with "= WGBS of Xu mouse SCNT and iPSC samples = == End repair == * Total Volume 70ul, sheared DNA 50ul, reaction mix 20ul {| {{table}} border = 1 | align="center" style="background...") |
>Zsakura2 |
||
(12 intermediate revisions by 2 users not shown) | |||
Line 1: | Line 1: | ||
= WGBS of Xu mouse SCNT and iPSC samples = | = WGBS of Xu mouse SCNT and iPSC samples = | ||
*sheared 3ug of gDNA for the samples below, and used 1.5 ug for WGBS sample prep | |||
{| {{table}} <TABLE BORDER="3" CELLPADDING="3" CELLSPACING="3"> style="text-align: center;" | |||
| align="center" style="background:#f0f0f0;"|'''sample ID''' | |||
| align="center" style="background:#f0f0f0;"|'''N2 Index used''' | |||
| align="center" style="background:#f0f0f0;"|'''cell type''' | |||
| align="center" style="background:#f0f0f0;"|'''progenitor line''' | |||
| align="center" style="background:#f0f0f0;"|'''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 == | == End repair == | ||
* Total Volume 70ul, sheared DNA 50ul, reaction mix 20ul | * Total Volume 70ul, sheared DNA 50ul, reaction mix 20ul | ||
Line 38: | Line 72: | ||
- E2-H2 ~31ul | - E2-H2 ~31ul | ||
= Adapter ligation = | == Adapter ligation == | ||
* Total volume 50ul, A-tail reaction mix 40ul, methylation adapters 10ul | * Total volume 50ul, A-tail reaction mix 40ul, methylation adapters 10ul | ||
{| {{table}} border = 1 | {| {{table}} border = 1 | ||
Line 62: | Line 96: | ||
- Note: We don't have enough adapter, so I reduced to 2ul | - Note: We don't have enough adapter, so I reduced to 2ul | ||
{| {{table}} border = 1 | {| {{table}} border = 1 | ||
| Prepare adapter|| | | Prepare adapter||3.0ulx 13rxn||39.00 | ||
|- | |- | ||
| H2O||7.0ulx13rxn||91.00 | | H2O||7.0ulx13rxn||91.00 | ||
|- | |- | ||
| Total|||| | | Total||||130.00 | ||
|} | |} | ||
- Aliquot to column #4: | - Aliquot to column #4: | ||
Line 72: | Line 106: | ||
- E4-H4 ~11ul | - E4-H4 ~11ul | ||
{| {{table}} <TABLE BORDER="3" CELLPADDING="3" CELLSPACING="3"> style="text-align: center;" | - 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 == | |||
Performed bisulfite conversion manually | |||
Elute with 40ul Elution buffer (final volume ~35ul) | |||
== Amplification of bisulfite sequencing libraries with N2 barcoded primers == | |||
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 == | |||
{| {{table}} <TABLE BORDER="3" CELLPADDING="3" CELLSPACING="3"> style="text-align: center;" | |||
| align="center" style="background:#f0f0f0;"|'''sample ID''' | | align="center" style="background:#f0f0f0;"|'''sample ID''' | ||
| align="center" style="background:#f0f0f0;"|'''N2 Index used)''' | | align="center" style="background:#f0f0f0;"|'''N2 Index used''' | ||
| align="center" style="background:#f0f0f0;"|'''amount needed for pooling (ul)''' | |||
|- | |- | ||
| 2A4F1||N2 index 1 | | 2A4F1||N2 index 1||5.08 | ||
|- | |- | ||
| 2A4F33||N2 index 2 | | 2A4F33||N2 index 2||7.05 | ||
|- | |- | ||
| miPS D1||N2 index 3 (not sequenced) | | miPS D1||N2 index 3 (not sequenced)||- | ||
|- | |- | ||
| miPS B3||N2 index 4 | | miPS B3||N2 index 4||7.77 | ||
|- | |- | ||
| 1E12P20||N2 index 5 | | 1E12P20||N2 index 5||7.43 | ||
|- | |- | ||
| nt ES P7 C||N2 index 6 | | nt ES P7 C||N2 index 6||12.5 | ||
|- | |- | ||
| nt ES P8 B||N2 index 7 | | nt ES P8 B||N2 index 7||33.17 | ||
|- | |- | ||
| NB 1||N2 index 8 (not sequenced) | | NB 1||N2 index 8 (not sequenced)||- | ||
|- | |- | ||
| NB 3||N2 index 9 | | NB 3||N2 index 9||4.95 | ||
|- | |- | ||
| SCNT B12||N2 index 10 | | SCNT B12||N2 index 10||4.88 | ||
|- | |- | ||
| B6 ES||N2 index 11 | | B6 ES||N2 index 11||4.88 | ||
|- | |- | ||
| 129 ES||N2 index 12 | | 129 ES||N2 index 12||6.75 | ||
|} | |} | ||
== Sequencing and read mapping summary== | |||
*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 | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Sample IDs''' | |||
| align="center" style="background:#f0f0f0;"|'''Experiment''' | |||
| align="center" style="background:#f0f0f0;"|'''Index number''' | |||
| align="center" style="background:#f0f0f0;"|''' Total reads''' | |||
| align="center" style="background:#f0f0f0;"|''' Mapped reads''' | |||
| align="center" style="background:#f0f0f0;"|'''Mapping rate''' | |||
| align="center" style="background:#f0f0f0;"|'''Conversion rate*''' | |||
| align="center" style="background:#f0f0f0;"|'''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 | |||
|- | |||
| | |||
|} | |||
[[Image:Hclusts-10kb-ZL-mSCNT-WGBS.png|400px]] |
Latest revision as of 01:40, 7 March 2014
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