GSM8012562: Mel_H3K4me3; Homo sapiens; ChIP-Seq
ChIP of H2AUb, H3K4me and H3K27me3 were performed from 6 millions of cells originating from Patient Derived Tumour grafts; magnetic Dynabeads coupled to Protein A were used for the IP (Invitrogen). Sonication was performed to obtain fragment size of 150-300 bp. Chip of H3K27Ac and CTCF were performed according the Diagneode protocols. The chromatin was prepared using the iDeal ChIP-seq Kit for Transcription Factors (Diagenode Cat# C01010170). Chromatin was sheared using Bioruptor® Pico sonication device (Diagenode Cat# B01060001) combined with the Bioruptor® Water cooler for 10 cycles using a 30'' [ON] 30'' [OFF] settings to obtain fragment size of 150-300 bp. Per H3K27Ac and CTCF ChIP,  2 millions of cells were prepared in 100μl. The library preparation has been conducted by Diagenode ChIP-seq/ChIP-qPCR Profiling service (Diagenode Cat# G02010000). Libraries were prepared using IP-Star® Compact Automated System (Diagenode Cat# B03000002) from input and ChIP'd DNA using MicroPlex Library Preparation Kit v3 (96 indexes) (Diagenode Cat# C05010004). 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 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 prepared accordingly to manufactures (TruSeq ChIP sample Prep Kit, Illumina). Sequencing was performed on a Illumina Hi-Seq 2500 (H2Aub, H3K4me3, H3k27me3) and on a Illumina NovaSeq (ChIPseq H3K27Ac, CTCF)