GSM4656267: DDIT3_DDIT3_2; Homo sapiens; ChIP-Seq
For chromatin immunoprecipitation, we used the iDeal ChIP-seq kit for Transcription Factors (Diagenode, cat No: C01010170). For cross-linking proteins with DNA, formaldehyde (37%) was mixed with Fixation Buffer (Diagenode), to a final concentration of 11% and added to the cultured cells medium in a proportion of 1:10, and incubated with gentle shaking at room temperature for 15 minutes. The reaction was stopped by adding glycine (1:10) for 5 minutes with gentle shaking at room temperature. The cells were collected by scraping and lysed with lysis buffers from Diagenode. chromatin was sheared by sonication using Bioruptor Pico sonication device (Diagenode, Belgium) (7 cycles, 30 seconds ON, 30 seconds OFF). Chip-seq grade antibodies (H3K27ac #C15410196, H3K27me3 #C15410195, CTCF #C15410210, Diagenode), (DDIT3 #2895, Cell Signaling Technology) mixed with magnetic beads (Diagenode) were added to 250 µl of sheared chromatin and incubated under constant rotation at 4 ⁰C overnight. A volume of 2.5 µl was treated in the same but without antibody and used as input. The beads were washed with washing buffers from (Diagenode) and elution buffer was added and samples were incubated overnight at 65 ⁰C. for DNA purification  IPure beads v2 (Diagenode) were used. The beads were washed with washing buffers from (Diagenode), then resuspended with buffer C (Diagenode) and kept for further analysis. Library preparation was performed by (Diagenode, Belgium). ChIP'd DNA was quantified using QuBit HS ds DNA kit. Libraries were prepared using IP-Star® Compact Automated System (Diagenode Cat# B03000002) from input and ChIP'd DNA using MicroPlex Library Preparation Kit v2 (12 indices) (Diagenode Cat# C05010013). Optimal library amplification was assessed by qPCR using KAPA SYBR® FAST (Sigma-Aldrich) on LightCycler® 96 System (Roche) and by using High Sensitivity NGS Fragment Analysis Kit (DNF-474) on a Fragment Analyzer™ (Agilent). Libraries were then purified (double-size selected) using Agencourt® AMPure® XP (Beckman Coulter) and quantified using Qubit™ dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32854). Finally their fragment size was analised by High Sensitivity NGS Fragment Analysis Kit (DNF-474) on a Fragment Analyzer™ (Agilent). Libraries were pooled and sequenced with Illumina technology with single-end reads of 50bp length. Sequencing was performed by (Diagenode, Belgium).