title: GSM4774132: LVshNONtg_H3K4me3_rep2; Homo sapiens; ChIP-Seq
library_construction_protocol: 72 hours following lentiviral transduction, cells were treated with 1% formaldehyde in cell culture media for 15 min at room temp. Formaldehyde was quenched by the addition of 0.125 M Glycine, and cells were washed with PBS. 4x10^6 crosslinked cells were used per IP. Chromatin from crosslinked cells was fractionated by digestion with micrococcal nuclease using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling) per the manufacturers' instructions. IPs were performed using the SimpleChIP Enzymatic Chromatin IP Kit per the manufacturers' instructions. 5 uL H3K4me3 (Cell Signaling, 9751) or 5 uL H3K27me3 (Cell Signaling, 9733) antibody was used per IP sample. DNA was purified using the ChIP DNA Clean & Concentrator Kit (Zymo). Libraries for Input and IP samples were prepared by the Van Andel Genomics Core from 10 ng of input material and IP material using the KAPA Hyper Prep Kit (v5.16) (Kapa Biosystems, Wilmington, MA USA). Prior to PCR amplification, end-repaired and A-tailed DNA fragments were ligated to Bioo Scientific NEXTflex Adapters (Bioo Scientific, Austin, TX, USA). Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.), QuantiFluor® dsDNA System (Promega Corp., Madison, WI, USA), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems). Individually indexed libraries were pooled and 75 bp, single-end sequencing was performed on an Illumina NextSeq 500 sequencer using 75 cycle HO sequencing kits (v2) (Illumina Inc., San Diego, CA, USA), with all libraries run across 2 flowcells to return a minimum read depth of 80M reads per input library and 40M read per IP library. Base calling was done by Illumina NextSeq Control Software (NCS) v2.0 and output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.