title: GSM2583730: H3K27me3_HiChIP_3; Drosophila melanogaster; OTHER
library_construction_protocol: 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-CP190 or anti-Pol2phosphoSerine2 antibody overnight. Isolated chromatin was washed, eluted, reverse crosslinked and purified by standard methods, after which ligation events were enriched by streptavidin precipitation. For ATAC-seq Kc167 cells grown to exponential stage were treated with DMSO or triptolide as previously described. 200,000 ctrl and treated cells were collected and used for Fast-ATAC protocol. Briefly, cell pellets were resuspened in 50 µl transposes Tn5 mixture (0.01% digitonin for permeabilizing cell membrane, 2.5 µl Tn5, 25 µl TD buffer), and incubated at 30°C for 20 min with occasional shaking. After reaction, cells were cooled on ice and continued with DNA extraction by Minelute Kit (Qiagen). Libraries were constructed using the standard protocol. Genomic fragments were end repaired (NEBNext End Repair Module), 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). Libraries were amplified with Illumina primers using the KAPA SYBR FAST qPCR Master Mix.
attributes.tag: GEO Accession
attributes.value: GSM2583730