Alice:Exome/2012-12-14

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Revision as of 01:40, 7 March 2014 by >Zsakura2 (→‎WGBS of Xu mouse SCNT and iPSC samples)
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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