GSM8086055: H3K27me3_RKO_Input; Homo sapiens; ChIP-Seq
Chromatin preparation: After the indicated treatments, RKO cells were crosslinked with 1% formaldehyde. The chromatin preparation for H3K9me3, UHRF1, and H3K27me3 ChIP were performed using sonication-based chromatin shearing as follows: About 10 million cells were resuspended in cell lysis buffer and nuclei were isolated and chromatin was fragmented with a Covaris E220 Evolution Focused-Ultrasonicator for 12 min with default settings. The chromatin preparation for H3K18ub ChIP was performed using MNase digestion as follows: About 10 million cells were resuspended in cell lysis buffer and nuclei were isolated and fragmented by 50U MNase (NEB). The digested chromatin was further solubilized with Covaris E220 Evolution Focused-Ultrasonicator for 1 min with default settings. IP: 5 ul H3K9me3 antibody (Active Motif,Cat#39161) and 5 ug of soluble chromatin, 2.5 ul UHRF1 antibody (Bioworld,Cat# BS7227) and 10 ug soluble chromatin, 5 ul H3K18ub antibody (Cell Signaling, Cat#37771) and 20 ug soluble chromatin, and 5 ul H3K27me3 antibody (Cell Signaling,Cat#9733) and 5 ug soluble chromatin were incubated at 4°C for 18 hours. Samples were immunoprecipitated with 30 ul Dynabeads Protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 2 hours followed by washes. IP chromatin was eluted and the eluted chromatin was subject to reverse crosslink and DNA purification before sequencing. Libraries for Input and IP samples were prepared by the Van Andel Genomics Core from 10 ng of input material and all available 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 uniquely barcoded dual indexes (IDT, Coralville, IA 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). 50 bp, paired-end sequencing was performed on an Illumina NovaSeq6000 sequencer using an S2, 100 bp sequencing kit (Illumina Inc., San Diego, CA, USA) to a minimum read depth of 50M read pairs per IP library and 100M read pairs per Input library. Base calling was done by Illumina RTA3 and the output of NCS was demultiplexed and converted to FastQ format with Illumina Bcl2fastq (v1.9.0).