GSM4979864: RRBS_somites_tif1g_KD_clutch8; Danio rerio; Bisulfite-Seq
15 (22 hpf) or 20 (11 hpf and 48 hpf) snap-frozen embryos were lysed in lysis buffer for 1 h at 55°C (10 mM Tris-HCl (pH=8.0), 10 mM NaCl, 10 mM EDTA, 0.5% SDS, 300 µg/ml proteinase K). Genomic DNA was isolated by phenol-chloroform extraction and quantified using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). RRBS was performed on 100 ng of each sample using the NuGen Ovation® RRBS Methyl-Seq System (Tecan, 0353) following the manufacturer's recommendations with the following modifications. After the final repair step, the bisulfite conversion of DNA was conducted using the EpiTect Fast Bisulfite Conversion Kit (Qiagen, 59824) following the manufacturer's recommendations, eluting the bisulfite-converted DNA in 23 µl EB. Libraries were amplified with 12 cycles of PCR. Amplified libraries were purified twice using RNAClean XP SPRI paramagnetic beads (Beckman Coulter, A63987) at 1x reaction volume. The purified libraries were quality-assessed with a D1000 ScreenTape (Agilent, 5067-5582) on the Agilent 4150 TapeStation System and sequenced for 100-bp single-end reads on the NovaSeq6000 (Illumina).