GSM8970087: HD3, DIF-mut55, UMI-4C, rep1; Gallus gallus; OTHER
Cells were fixed in 1% paraformaldehyde and quenched with 0.125M Glycine. Cell were lysed 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. 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.