GSM5351648: UMI-4C in zebrafish wt embryos at 48hpf, hoxa5a promoter as viewpoint; Danio rerio; OTHER
Embryos were fixed in 1% paraformaldehyde and quenched with 0.125M Glycine. Cell were lyses and extracted nuclei were processed for in-situ HiC preparation as previously described (Rao et al. 2014). Briefly, chromatin was digested with DpnII, fragment-ends were blunted and marked with dATP-biotin, followed by proximity ligation. DNA was extracted after cross-link reversal and sonicated in a Covaris M220 sonicator for targeted fragmentation with an average peak size at 500 bp. Proximity ligation products were enriched using Streptavidin-coated beads. End-repair and A-tailing was performed following standard procedure for Illumina library preparations for paired-end sequencing. 5'-DNA ends were then dephosphorylated using Alkaline Phosphatase, Calf Intestinal. Libraries were then indexed by ligation of TruSeq indexing adapters from Illumina. Non-ligated strand from adapter was removed by denaturation of DNA at 96°C. Final library was prepared using NEBNext High-Fidelity 2X PCR Master Mix (NEB) and ligation junctions for specific viewpoints were enriched using a nested PCR approach as described in Schwartzman et al. 2016. Viewpoint-specific downstream (DS) primers contained P5 sequence at their 5' end and TruSeq Primer 2.0 (P7) was used to complement adapter sequences for pair-end sequencing.