title: GSM4852393: CP190_PITA_delta_repl2 ChIP-seq; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: Chromatin for input (0.5 ug of DNA) was brought to RIPA conditions (140mM NaCl / 10mM Tris-HCl pH8,0 / 1mM EDTA / 1% TritonX100 / 0,1% SDS / 0,1% sodium deoxycholate, 1mM PMSF) was diluted in TE with 0.5% SDS, treated with RNase A for 30 min and protease K overnight, incubated at 65 degrees for 6 hours and phenol chloroform extracted. Sepharose was resuspended in TE with 0.5% SDS, treated with RNase A for 30 min and protease K overnight, incubated at 65 degrees for 6 hours and phenol chloroform extracted. Libraries were prepared according to manufacturers recommendations with small modifications. In short, 1-10ng of purified, RNase treated, and reverse cross-linked genomic DNA was end-repaired and terminal adenosine residues were added using the NEBNext reagents. Custom-made indexed adapters were ligated, after which the material was size selected at ~200-600 bp with Ampure XP beads (Beckman Coulter). PCR amplification was performed using PE1.0 and PE2.0 primers (custom-made) for 12 cycles for Input samples and 14 to 15 cycles for IP-ed samples using the Q5 Hot Start HiFi PCR Master Mix (NEB). The PCR-amplified library was purified using Ampure XP beads and its quality was assessed on a Bioanalyzer 2100 system (Agilent). The libraries were sequenced on a HiSeq 2000 (Illumina) in single end mode.