2025-08-08
ENA first public
ERC000011
ENA-CHECKLIST
Nuclear Dynamics Unit UMR3664 CNRS/IC Institut Curie - Centre de Recherche
INSDC center name
public
INSDC status
ERS20948818
SRA accession
E-MTAB-14416:KO_H31SNAP_input
Submitter Id
ArrayExpress
broker name
HeLa
cell_line
epithelial cell
cell_type
not collected
collection date
human
common name
Protocols: We collected 5 million cells per IP by trypsinization. We used HeLa cells stably expressing H3.1-SNAP-HA or H3.3-SNAP-HA which were either wild-type (WT, CRISPR/Cas9 GFP KO) or knock-out for HIRA (KO, CRISPR/Cas9 HIRA KO), as described in Ray-Gallet et al. (2018). We cultured cells in DMEM complete medium (Dulbecco’s Modified Eagle’s Medium with D-Glucose, L-Glutamine and Pyruvate) supplemented with 10% fetal calf serum, 100U/mL Penicillin and 100mg/mL Streptomycin.  We performed ChIP-seq using the native nucleosomes isolation procedure described in Gatto et al. (2022) with small modifications. We used 5 million cells per IP and Dynabeads Protein A-conjugated antibodies against histone PTMs for immunoprecipitation. All steps were performed at 4°C and in the presence of Protease inhibitors (Roche) and 1mM TSA in every buffer to prevent protease and HDAC activity, respectively. For each IP reaction, we prepared 50μL of Ab-conjugated beads by blocking for 4 hours in Bead blocking buffer (2.5% BSA in PBS-T, 1mg/mL tRNA), washing once in 0.02% PBS-T and incubating for 15-30min with the antibodies (diluted in 0.2mL 0.02% PBS-T) on a rotating wheel. Ab-conjugated beads were then washed twice with 0.02% PBS-T and resuspended in 0.2mL 0.02% PBS-T. Native nucleosomes (80μL per IP) from up to 3 samples were pooled, diluted in 5x volumes of Incubation buffer (50mM Tris-HCl pH 7.5, 100mM NaCl, 0.5% BSA) and pre-cleared by incubating with Dynabeads Protein A (15μL per input pool) for 30 min on a rotating wheel. We kept 20μL (1%) pre-cleared chromatin as input sample. We incubated the remaining (460μL/IP) with the Ab-conjugated beads (washed once in Incubation buffer) overnight on a rotating wheel. Beads were washed and DNA was purified from the IPed material as described for SNAP-seq in Gatto et al. (2022).  Ab used: PTM     Provider        Cat. no Amount per ChIP (ug) H3K4me1 abcam         ab8895 4 H3K4me3 Active Motif 39915 4 H3K9me3 Active Motif 39765 10 H3K27ac Active Motif 39133 5 H3K7me3 Active Motif 39155 5  Sequencing libraries were prepared at the NGS (Next-Generation Sequencing) platform at Institut Curie with the Illumina TruSeq ChIP kit.
description
adult
developmental stage
cervical adenocarcinoma
disease
H3.1-SNAP-HA HIRA CRISPR/Cas9 KO
genotype
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
uterine cervix
organism part
Homo sapiens
scientific_name
KO_H31SNAP_input
title