title: Bisulfite sequencing of primary keratinocyte_3
design: The BisPCR2 method including bisulfite conversion of genomic DNA and two rounds of PCR for target enrichment and sample barcoding was performed according to a previous study with minor modifications [1]. Specifically, hippocampal tissues were incubated with tissue lysis buffer ( 10 mM Tris pH 8.0, 100 mM NaCl, 10 mM EDTA pH 8.0, 10% SDS) containing proteinase K at 56overnight, and DNA was extracted by phenol-chloroform and quantified by NanoDrop2000. The genomic DNA was bisulfite converted using EZ DNA Methylation-Gold Kit (ZYMO) according to the manufacturers instruction. The bisulfite-converted DNA was amplified with primers specific for the RELA gene promoter region. The first round PCR was performed in a 25-l reaction containing 0.1 M methylation specific primers (see table), 100 ng Bisulfite converted DNA, 5 U of EpiTaq HS DNA polymerase (Takara), 200 M of each dNTP, and 1 reaction buffer. The first round of PCR was performed at 98 C for 30 s, 26 cycles of 95 C for 10 s, 50C for 30s,72C for 30s, and finally 72C for 5min.The PCR products were pooled together and purified with 1.2 AMPure XP beads (Beckman Coulter) to remove primer dimers. The second round PCR was performed in a 25-l reaction containing 0.1 M sample index primers, 10ng pooled PCR products, 5 U of Phusion DNA polymerase (NEB), 200 M of each dNTP, and 1 reaction buffer. The second round of PCR was performed at 95 C for 30 s,10 cycles of 95C for 15s, 59C for 30s,72C for 30s, and finally 72 C for 5 min (sample index primers are listed in table). Barcoded libraries were pooled together and purified with 1.5X AMPure XP beads (Beckman Coulter, Brea, CA) , and then were sent for sequencing of 2 150-bp paired-end reads on Illumina Nova-seq 6000 (San Diego, CA).