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
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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||2.0ulx 13rxn||26.00
| Prepare adapter||3.0ulx 13rxn||39.00
|-
|-
| H2O||7.0ulx13rxn||91.00
| H2O||7.0ulx13rxn||91.00
|-
|-
| Total||||117.00
| 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