GSM3564261: H3K36me3 ChIP-seq in 22Rv1 rep1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Histone
Antigen
H3K36me3
Cell type
Cell type Class
Prostate
Cell type
22Rv1
Primary Tissue
Prostate
Tissue Diagnosis
Carcinoma
Attributes by original data submitter
Sample
source_name
H3K36me3 ChIP-seq in 22Rv1
cell line
22Rv1
cell type
Human prostate cancer cell line
chip antibody
H3K36me3
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
In situ Hi-C experiments were performed following the original protocol by Rao et al with minor modifications. In situ Hi-C was performed in duplicate and 5 x 106 cells were used for each experiment. 100U of MboI restriction enzyme (NEB, R0147) was used to digest chromatin. For ligation, 2000U T4 DNA Ligase (NEB, M0202) was added and incubated at room temperature for 4 hours with slow rotation. Hi-C material was sheared to a size of 300-500bp using a Covaris instrument (Covaris S2, Woburn, MA). Biotin-tagged DNA was pulled down using Dynabeads MyOne Streptavidin C1 beads (Life technologies, 65002) with 2X Binding Buffer (2X BB: 10mM Tris-HCl (pH 7.5), 1nM EDTA, 2M NaCl). ChIP assays were performed in C42B, 22Rv1 and RWPE1 cells using H3K9me3 (Cat# 13969 Lot# 1, Cell Signaling and Technology, Inc.), H3K27me3 (Cat# 9733 Lot# 8, Cell Signaling and Technology, Inc.), and H3K36me3 (Cat# 2901 Lot# 3, Cell Signaling and Technology, Inc.) antibodies, according to ENCODE standards (https://www.encodeproject.org/data-standards/). RNA-seq was performed in triplicate for RWPE1, C42B, and 22Rv1 cells. RNA was extracted using Trizol reagent (Cat # 15596-018, Thermo Fisher Scientific, NY, USA) and the quality of RNA was assessed using a 2100 Bioanalyzer instrument (Cat # G2939AA, Agilent technologies), as described previously. For NOMe-seq, after isolating nuclei from the cells, M.CviPI was treated to methylate accessible GpCs. After purifying M.CvPI-treated DNA, sonication was performed. Bisulfite treatment of M.CviPI-methylated DNA resulted to convert all unmethylated Cs to Ts. In situ Hi-C ibraries were prepared using Kapa Hyper prep kit (Kapa #KK8503) according to the provided protocol. Library was amplified with PCR using Illumina primers. ChIP-seq: Libraries were barcoded (NEXTflex™ DNA Barcodes) (Bio Scientific, Austin, TX). RNA-seq libraries were made using KAPA Stranded mRNA-Seq Kit with KAPA mRNA Capture Beads (KK8420) (Kapa Biosystems, Woburn, MA). NOMe-seq: Libraries were generated using the Accel-NGS Methyl-Seq DNA Library Kit for Illumina Platforms.