Alice:Exome/2012-12-14: Difference between revisions
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>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 No edit summary |
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= 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)''' | |||
|- | |||
| 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 | |||
|} | |||
== 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 69: | ||
- 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 93: | ||
- 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 103: | ||
- 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 | ||
|} | |} |
Revision as of 23:33, 22 January 2013
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
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 |
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
- 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
- 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 | 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
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
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 |