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= 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 == * Total Volume 70ul, sheared DNA 50ul, reaction mix 20ul {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''NEB End repair mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''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 {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''A-tailing reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''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 {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Ligation reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''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 {| {{table}} border = 1 | 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 == {| {{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;"|'''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== *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]]
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