Bisulfite-seq of Homo sapiens: fetal sacral fibroblast cell line
Cells were FACS sorted into 3uL of freshly prepared Zymo EZ DNA Methylation-Gold CT Conversion Reagent, then heated [98C 8m, 3x(64C 1h, 98C 2m)]. 1uL of 10M sodium hydroxide was added; the reaction was then incubated at 37C 15m and 96 uL of 10.4 mM Tris-HCl was added. 14 uL first strand synthesis mix (170 uL first strand primer mix [0.5 M Oligo 1 and 0.625 nM each G-poor CpG spike-in primer], 170 uL 10 mM dNTPs, 1166 uL 10x Blue Buffer) was added, heated to 65C 3m, then chilled to 15C. 1 uL (50U) of high-concentration klenow 3-5 exo- polymerase (Qiagen) was added, and a linear amplification incubation was initiated (15C 5m, slow heating [0.1C/sec] to 37C, hold 5m, 15C 10s, 95C 1m, 15C hold). 2.5 uL of master mix (740 uL first strand primer mix, 74 uL 10 mM dNTPs, 185 uL 10x Blue Buffer, 370 uL high-concentration klenow exo-, 481 uL water) was added and another linear amplification incubation was performed. This process was repeated 5 times. Finally, 5 uL of master mix was added and a final linear amplification incubation was performed (15C 5m, slow heat (0.1C/sec) to 37C, hold 37C 90m, 4C hold). In this way, a total of seven rounds of random priming were completed. Prior to second strand synthesis, 2 uL (40 U) Exo I (NEB) was added, and the reaction was incubated at 37C 1h. A 0.8x ratio bead cleanup (Beckman Coulter SPRIselect) was then performed, eluting into 40 uL of water. 9 uL of second-strand master mix (206 uL 10 mM dNTP, 206 uL second strand primer mix [10 M Oligo 2 and 12.5 nM each C-poor CpG spike-in primer], 515 uL 10x Blue buffer) was added, and the reaction was incubated at 95C 1m, then cooled to 4C. 2 uL (100U) of klenow exo- was added, and the reaction was incubated at 4C 5m, slowly heated (0.1C/sec) to 37C, held at 37C 90m, then cooled to 4C. A second 0.8x ratio bead cleanup was performed, eluting into 23 uL of water. 23 uL of KAPA HotStart ReadyMix master mix (Roche) and 2 uL of xGen UDI indexing primers (IDT) were added, followed by PCR amplification (95C 2m; 12-15x(94C 80s, 62C 30s, 72C 40s); 72C 3m). An 0.7x ratio bead cleanup was performed eluting into 21 uL of 10 mM low-EDTA Tris.