GSM3346512: RNAPII ChIP-seq Rad21 KD; Drosophila melanogaster; ChIP-Seq
Cells were crosslinked with 1% formaldehyde for 10 min at room temperature and quenched with glycine. For ChIP-seq, nuclear lysates were sonicated to generate 200-500 bp fragments . Chromatin was precleared with Protein A or G Dynabeads at 4°C for 2 hours and incubated with antibody overnight at 4°C. Isolated chromatin was washed, eluted, reverse crosslinked and purified by standard methods. For HiChIP nuclei were isolated and chromatin digested by DpnII, filled in with biotin-dCTP, and ligated. After ligation, chromatin was sonicated and precleared with Protein A and G Dynabeads at 4°C for 2 hours, then precipitated using anti-RNAPII or anti-Rad21 antibody overnight. Isolated chromatin was washed, eluted, reverse crosslinked and purified by standard methods, after which ligation events were enriched by streptavidin precipitation. Libraries were constructed using the standard protocol for ChIP-seq and Hi-C or using a Tn5 library prepartion protocol for HiChIP. Genomic fragments were end repaired, A-tailed by adding  adenosine to  the 3' ends of fragment using Klenow fragment (3' to 5' exo minus, New England Biolabs), and adaptors were ligatedat room temperature for 1 hr with T4 DNA ligase (New England Biolabs). For Tn5 protocol samples were incubated with Tn5 transposase in the presence of adaptors. Libraries were amplified with Illumina primers using the KAPA SYBR FAST qPCR Master Mix.