Antigen

Antigen Class

Histone

Antigen

H4K20me1

Cell type

Cell type Class

Prostate

Cell type

Prostate

MeSH Description

A gland in males that surrounds the neck of the URINARY BLADDER and the URETHRA. It secretes a substance that liquefies coagulated semen. It is situated in the pelvic cavity behind the lower part of the PUBIC SYMPHYSIS, above the deep layer of the triangular ligament, and rests upon the RECTUM.

Sample

source_name

WT ventral prostates

cell line

ventral prostatic tissues

genotype/variation

wild type

antibody

anti-H4K20me1

antibody manufacturer

Abcam

antibody catalog number

#ab9051

diet

CTD

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Fresh-frozen VP tissues from 12-week-old mice were pulverized (Cryoprep Impactor, Covaris), resuspended in PBS + 1% formaldehyde, and incubated at room temperature for 20 minutes. Fixation was stopped by the addition of 0.125 M glycine (final concentration) for 15 minutes at room temperature, then washing in ice cold PBS + EDTA-free protease inhibitor cocktail (PIC; #04693132001, Roche). Multiple biological replicates were combined for each condition in two distinct pools (replicates). Chromatin was isolated by the addition of lysis buffer (0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA (pH 8.0), 0.1% NaDOC, 0.13 M NaCl, 1X PIC) + sonication buffer (0.25% sarkosyl, 1 mM DTT) to the samples, which were maintained on ice for 30 minutes. Lysates were sonicated (E210 Focused-ultrasonicator, Covaris) and the DNA was sheared to an average length of ~200-500 bp. Genomic DNA (input) was isolated by treating sheared chromatin samples with RNase (30 minutes at 37°C), proteinase K (30 minutes at 55°C), de-crosslinking buffer (1% SDS, 100 mM NaHCO3 (final concentration), 6-16 hours at 65°C), followed by purification (#28008, Qiagen). DNA was quantified on a NanoDrop spectrophotometer, using the Quant-iT High-Sensitivity dsDNA Assay Kit (#Q33120, Thermo Fisher Scientific). On ice, ChIP-validated H4K20me1 (2 micrograms, #ab9051, Abcam) or PHF8 (5 micrograms, #A301-772A, Bethyl Laboratories) antibodies were conjugated to a mix of washed Dynalbeads protein A and G (Thermo Fisher Scientific), and incubated on a rotator (overnight at 4°C) with 1.5 micrograms (H4K20me1) or 5 micrograms (PHF8) of chromatin. ChIP’ed complexes were washed, sequentially treated with RNase (30 minutes at 37°C), proteinase K (30 minutes at 55°C), de-crosslinking buffer (1% SDS, 100 mM NaHCO3 (final concentration), 6-16 hours at 65°C), and purified (#28008, Qiagen). The concentration and size distribution of the immunoprecipitated DNA was measured using the Bioanalyzer High Sensitivity DNA kit (#5067-4626, Agilent). Dana-Farber Cancer Institute Molecular Biology Core Facilities prepared libraries from 2 ng of DNA, using the ThruPLEX DNA-seq kit (#R400427, Rubicon Genomics), according to the manufacturer’s protocol; finished libraries were quantified by the Qubit dsDNA High-Sensitivity Assay Kit (#32854, Thermo Fisher Scientific), by an Agilent TapeStation 2200 system using D1000 ScreenTape (# 5067-5582, Agilent), and by RT-qPCR using the KAPA library quantification kit (# KK4835, Kapa Biosystems), according to the manufacturers’ protocols; ChIP-seq libraries were uniquely indexed in equimolar ratios, and sequenced to a target depth of 40M reads on an Illumina NextSeq500 run, with single-end 75bp reads.

Sequencing Platform

instrument_model

NextSeq 500

mm10

Number of total reads

145934478

Reads aligned (%)

90.8

Duplicates removed (%)

19.4

Number of peaks

1191 (qval < 1E-05)

mm9

Number of total reads

145934478

Reads aligned (%)

90.7

Duplicates removed (%)

19.4

Number of peaks

1201 (qval < 1E-05)