Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
Pcgf1

Cell type

Cell type Class
Pluripotent stem cell
Cell type
ES cells
NA
NA

Attributes by original data submitter

Sample

source_name
Mouse embryonic stem cells with human HEK293T cells spike-in, tamoxifen-treated (72hr OHT), PCGF1 ChIP-seq
cell line
PRC1CPM
genotype
Ring1aI50A/D53K; Ring1b(WT->I53A/D56K)fl/fl; Rosa26::ERT2-Cre
clone
41
strain
E14
replicate
3
treatment agent
tamoxifen (OHT)
treatment time point
72 hr
spike-in reference organism
Homo sapiens
spike-in cell line
HEK293T
ChIP
PCGF1
antibody
In house (Blackledge et al., 2014)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
For RING1B, SUZ12, JARID2, PCL2, PCGF2, PCGF1 and PCGF6, cChIP-seq was performed as described previously (Fursova et al., 2019). Briefly, 5 x 10^7 mouse ESCs (untreated or following 72 hours OHT treatment) were mixed with 2 x 10^6 human HEK293T cells. Cells were resuspended in 10 ml phosphate buffered saline (PBS) and crosslinked at 25°C with 2 mM DSG (Thermo Scientific) for 45 minutes, and then with 1% formaldehyde (methanol-free, Thermo Scientific) for a further 15 minutes. Reactions were quenched with 125 mM glycine. Crosslinked cells were incubated in lysis buffer (50 mM HEPES pH 7.9, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton-X100) for 10 minutes at 4˚C. Released nuclei were washed (10 mM Tris-HCl pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) for 5 minutes at 4˚C. Chromatin was then resuspended in 1 ml of sonication buffer (10 mM Tris-HCl pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na deoxycholate, 0.5% N-lauroylsarcosine) and sonicated for 30 minutes using a BioRuptor Pico (Diagenode), shearing genomic DNA to an average size of 0.5 kb. Following sonication, TritonX-100 was added to a final concentration of 1%. For ChIP, sonicated chromatin was diluted 10-fold in ChIP dilution buffer (1% Triton-X100, 1 mM EDTA, 20 mM Tris-HCl pH 8, 150 mM NaCl) and pre-cleared for 1 hour using Protein A agarose beads (Repligen) blocked with 1 mg/ml BSA and 1 mg/ml yeast tRNA. For each ChIP reaction, 1ml of diluted and pre-cleared chromatin was incubated overnight with the appropriate antibody, anti-RING1B (CST, D22F2, 3 ul), anti-SUZ12 (CST, D39F6, 3 ul), anti-PCGF1 (in-house, 5 ul), anti-PCGF2 (Santa Cruz, sc-10744, 3 ul), anti-JARID2 (CST D6M9X, 3 ul), anti-PCL2 (GenWay GWB-FA7207, 2 ul), or anti-PCGF6 (3 ul). Antibody-bound chromatin was captured using blocked protein A agarose for 1 hour at 4°C and collected by centrifugation. ChIP washes were performed as described previously (Farcas et al., 2012). ChIP DNA was eluted in elution buffer (1% SDS, 0.1 M NaHCO3) and cross-links were reversed overnight at 65oC with 200 mM NaCl and 2 ul RNase A (Sigma). A matched input sample (10% of original ChIP reaction) was identically treated. The following day, ChIP samples and Inputs were incubated with Proteinase K (Sigma) for 1.5 hours at 56oC and purified using ChIP DNA Clean and Concentrator Kit (Zymo Research). Native cChIP-seq for H2AK119ub1, H3K27me3, and H3K4me3 was performed as described previously (Fursova et al., 2019). Briefly, 5 x 10^7 mouse ESCs (both untreated and following 72 hours OHT treatment) were mixed with 2 x 10^7 Drosophila SG4 cells in PBS. Mixed cells were pelleted and nuclei were released by resuspending in ice cold lysis buffer (10mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 5 mM N-ethylmaleimide). Nuclei were then washed, and resuspended in 1 ml of MNase digestion buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.25 M sucrose, 3 mM CaCl2, 10 mM N-ethylmaleimide, 1x protease inhibitor cocktail (Roche)). Each sample was incubated with 200 units of MNase (Fermentas) at 37°C for 5 min, followed by the addition of 4 mM EDTA to halt MNase digestion. Following centrifugation at 1500 g for 5 min at 4°C, the supernatant (S1) was retained. The remaining pellet was incubated with 300 µl of nucleosome release buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2 mM EDTA, 1x protease inhibitor cocktail (Roche), 10 mM N-ethylmaleimide) at 4°C for 1 h, passed five times through a 27G needle using a 1 ml syringe, and spun at 1500 g for 5 min at 4°C. The second supernatant (S2) was collected and combined with corresponding S1 sample from above. A small amount of S1/S2 DNA was purified and visualized on a 1.5% agarose gel to confirm digestion to mostly mono-nucleosomes. For ChIP experiments, S1/S2 nucleosomes were diluted 10-fold in native ChIP incubation buffer (70 mM NaCl, 10 mM Tris pH 7.5, 2 mM MgCl2, 2 mM EDTA, 0.1% Triton, 1x protease inhibitor cocktail (Roche), 10 mM N-ethylmaleimide (NEM)), and 1 ml aliquots were made. Each ChIP reaction was then incubated overnight at 4oC with 5 ul of anti-H2AK119ub1 (CST D27C4), 5 ul of anti-H3K27me3 (in-house), or 3 ul anti-H3K4me3 (in-house) antibody. Antibody-bound nucleosomes were captured using protein A agarose (Repligen) beads, pre-blocked in native ChIP incubation buffer supplemented with 1 mg/ml BSA and 1 mg/ml yeast tRNA, for 1 hour at 4°C and collected by centrifugation. Immunoprecipitated material was washed four times with Native ChIP wash buffer (20 mM Tris pH 7.5, 2 mM EDTA, 125 mM NaCl, 0.1% Triton-X100) and once with Tris-EDTA buffer (10 mM Tris pH 8, 1 mM EDTA). ChIP DNA was eluted using 100 ul of elution buffer (1% SDS, 0.1 M NaHCO3), and then purified using ChIP DNA Clean and Concentrator Kit (Zymo Research). For each individual ChIP sample, DNA from a matched Input control (corresponding to 10% of original ChIP reaction) was also purified. cChIP-seq libraries for both ChIP and Input samples were prepared using NEBNext Ultra DNA Library Prep Kit for Illumina, following manufacturer's guidelines. Samples were indexed using NEBNext Multiplex Oligos. The average size and concentration of all libraries was analysed using the 2100 Bioanalyzer High Sensitivity DNA Kit (Agilent) followed by qPCR using SensiMix SYBR (Bioline, UK) and KAPA Illumina DNA standards (Roche). Libraries were sequenced as 40 bp paired-end reads on Illumina NextSeq 500 platform.

Sequencing Platform

instrument_model
NextSeq 500

mm10

Number of total reads
27242406
Reads aligned (%)
94.2
Duplicates removed (%)
5.7
Number of peaks
12101 (qval < 1E-05)

mm9

Number of total reads
27242406
Reads aligned (%)
94.0
Duplicates removed (%)
6.1
Number of peaks
12098 (qval < 1E-05)

Base call quality data from DBCLS SRA