title: Illumina HiSeq 2500 sequencing; Reduced representation bisulfite sequencing (RRBS) for primordial germ cells and somatic cells during zebrafish early embryogenesis
design: Reduced representation bisulfite sequencing (RRBS) for primordial germ cells and somatic cells during zebrafish early embryogenesis
library_construction_protocol: PGCs and somatic cells were isolated from Buc-GFP zebrafish strain at 3.3hpf, 4.3hpf and 24hpf via FACS. Cells were collected directly into 150ul 1.5X lysis buffer and snap frozen. Thaw cells and incubate with 20ug/ml RNase A at 37C for 1h, followed by 100ug/ml Proteinase K treatment at 55C for 3h. Phenol:choroform extraction and ethanol precipitation were performed to extract DNA and then resuspend it in water. Genomic DNA were extracted from 4500-7500 sorted somatic cells or PGCs at high, dome and 24hpf stage and digested with MspI at 37C for 3h. The fragment ends were repaired with Klenow exo at 37C for 50min. Then Methylated illumina Pair-end Adaptors were ligated to gDNA fragments using T4 DNA ligase. The bisulfite conversion were performed using Zymo Research EZ DNA Methylation Gold kit. Libraries were PCR amplified for 20 cycles using Platinum Taq DNA polymerase.