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== Amplification of bisulfite sequencing libraries with N2 barcoded primers == * Perform in 50ul reaction with PCR_R.Ind1-10, add 7ul of adapter-ligated bis-cvt DNA {| {{table}} | Width="200pt" align="center" style="background:#f0f0f0;"|'''Components''' | width="50pt" align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''11.5 rxn mix''' |- | Adapter ligated bis-cvt DNA||7.00||0.00 |- | 10uM PCR_F||1.00||11.50 |- | 10uM PCR_R.Ind1-8, 21,22||1.00||0.00 |- | 2x iQ MM||25.00||287.50 |- | H2O||16.00||184.00 |- | Total||50.00||483.00 |} * Aliquot 42ul, add 1ul PCR_R.IndX and 7ul of adapter ligated bis-cvt DNA '''Program''' 98C 2min --> (98C 15sec -> 64C 20sec -> 72C 45sec) x 10 cycles --> 72C 3min --> 15C Hold '''qPCR result''' [[File:20120827_qPCR_WGBS_N37_50ul.png| 600px]] * Performed qPCR in total volume 100ul by using the same condition as above (in duplicated) {| {{table}} | Width="200pt" align="center" style="background:#f0f0f0;"|'''Components''' | width="50pt" align="center" style="background:#f0f0f0;"|'''1rxn''' | align="center" style="background:#f0f0f0;"|'''21.5 rxn mix''' |- | Adapter ligated bis-cvt DNA||14.00||0.00 |- | 10uM PCR_F||2.00||43.00 |- | 10uM PCR_R.Ind1-8,21,22||2.00||0.00 |- | 2x iQ MM||50.00||1075.00 |- | H2O||32.00||688.00 |- | Total||100.00||1806.00 |} * Aliquot 84ul, add 2ul PCR_R.IndX and 14ul of adapter ligated bis-cvt DNA '''Program''' 98C 2min --> (98C 15sec -> 64C 20sec -> 72C 45sec) x 10 cycles --> 72C 3min --> 15C Hold '''qPCR results''' [[File:20120827_qPCR_WGBS_N37_rep1-2.png| 600px]] {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Sample IDs''' | align="center" style="background:#f0f0f0;"|'''Labeling''' | align="center" style="background:#f0f0f0;"|'''Indx''' |- | N37-Cerebellum (CRBL)||N37-1||PCR_R.N2Indx1 |- | N37-Colon||N37-2||PCR_R.N2Indx2 |- | N37-Frontal lobe (FL)||N37-3||PCR_R.N2Indx3 |- | N37-Heart||N37-4||PCR_R.N2Indx4 |- | N37-Small intestine (SI)||N37-5||PCR_R.N2Indx5 |- | N37-Liver||N37-6||PCR_R.N2Indx6 |- | N37-Lung||N37-7||PCR_R.N2Indx7 |- | N37-Skeletal muscle (SM)||N37-8||PCR_R.N2Indx8 |- | N37-Pancreas||N37-9||PCR_R.N2Indx21 |- | N37-Stomach||N37-10||PCR_R.N2Indx22 |} - Purify all PCR products (total volume 250ul) with 0.8 volume of 3x diluted AMPure bead by EPmotion - Elute with 70ul EB buffer - Perform PAGE quantification by loading 1ul of AMPure bead purified DNA [[File:ZhangLab_2 2012-08-27 17hr 22min_PQ_bead_WGBS_N37.jpg| 500px]]
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