Genome-wide chromatin accessibility and DNA methylation of wildtype zebrafish livers at 120 hpf

The goal of this study is genomic chromatin accessibility of 120 hpf zebrafish livers by ATAC-Seq. ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) and the DNA methylation profile using reduced representation bisulfite sequencing (RRBS). Overall design: "To perform ATAC-seq on pools of zebrafish larvael livers collected at 120 hpf, we microdissected between 10-20 livers from 3 independent clutches of Tg(fabp10a:CAAX-EGFP) following immobilization using tricaine. ATAC-seq was performed using the protocol described in Buenrostro et al (2015): doi:10.1002/0471142727.mb2129s109, with the exception of dissociating the livers for cell suspension was obtained by treatment with trypsin at 37 degree for 30 minutes. ATAC-seq: Protocol as described in Buenrostro et al (2015): doi:10.1002/0471142727.mb2129s109, using illumina barcoded primers for sequencing. To perform RRBS on WT zebrafish livers to determine which genomic regions are methylated/unmethylated specifically in this tissue. We dissected 40 livers from a different set of larvae tahn were used for ATACseq. These were WT siblings of a transgenic line following immobilization using tricaine at 5 days post fertilizaion (dpf). Livers were pooled and high molecular weight DNA from larvae was extracted by using a DNA extraction buffer as previously described (Chernyavskaya et al., 2017). DNA was used to perform RRBS library prep according to Garrett-Bakelman et al., 2015, with some modification. To avoid loss of gDNA, after MspI digestion, end repair, and Atailing, the reactions were stopped by heat inactivation. The adaptors used for multiplexing were purchased separately (Next Multiplex Methylated Adaptors–New England Biolabs) and libraries were size selected by dual-step purification with Ampure XP Magnetic Beads (Beckman Coulter, Agencourt).