|
| Status |
Public on Mar 29, 2022 |
| Title |
ChIPseq_H3K9me3_CT_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
A549 immortalized cell line
|
| Organism |
Homo sapiens |
| Characteristics |
antibody: H3K9me3
cell line: A549
genotype: Control (CT)
disease: lung adenocarcinoma
|
| Treatment protocol |
SETDB1 knockout was performed through CRISPR/Cas9-mediated gene disruption. The sgRNA were synthesized (Sigma) as oligonucleotide DNA sequences and cloned into PX458 plasmid (Addgene, Plasmid #48138). Cloning was performed according to a protocol described by Ran et al. (PMID: 24157548).
|
| Growth protocol |
A549 (K‐RAS G12S) cells were obtained from ATCC (ATCC CCL-185). Cells were cultured in Dulbecco’s modified Eagle’s Medium DMEM (Sigma) supplemented with 10% FBS (Gibco), 1% penicillin/streptomycin (Sigma), maintained at 37°C and 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA-seq: RNA extraction was performed using RNeasy mini-kit (Qiagen) followed by Turbo DNA-free kit (Ambion). ChIP-seq: Cells were collected with or without formaline fixation. Chromatin was fragmented by MNase digestion (histone ChIP) or sonication (CTCF ChIP), and immunoprecipitated by different antibodies. DNA was purified using MinElute columns from Qiagen. Hi-C: Cells were fixed with 2% of formaldehyde, chromatin was digested with DpnII, DNA ends were biotinylated and re-ligated in situ. DNA was purified by reverse-crosslinking, followed by phenol/chloroform extraction, ethanol precipitation and additional purification using AMPure XP magnetic beads. DNA was sheared by sonication, ligation junctions were enriched by Biotin pull-down.
RNA-seq: Libraries were constructed using Truseq stranded total RNA kit (Illumina). ChIP-seq: Libraries were prepared using the MicroPlex V2 library preparation kit (Diagenode). Hi-C: Libraries were prepared with standard protocol using TruSeq adapters (Illumina).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
input: Input_histone_CT_rep1
H3K9me3_CT.bw
H3K9me3_CT_peaks.bed
|
| Data processing |
RNA-seq: RNA-seq reads were mapped using STAR v2.6.1c with GENCODE v33 gene annotation. Read counts per gene were obtained using the ‘--quantMode’ parameter in STAR. Differentially expression analysis was performed with edgeR v3.30.0. Quasi-likelihood negative binomial generalized log-linear model and t-test relative to a fold change threshold of 1.5 were used to calculate per gene p-values. The obtained p-values were adjusted with Benjamini–Hochberg correction for multiple testing (FDR). FDR threshold of 0.01 was used to define differentially expressed genes.
ChIP-seq: ChIP-seq reads were mapped using bowtie v2.2.3 with ‘--no-discordant’ and ‘--no-mixed’ options. Uniquely mapped reads with MAPQ>30 were selected using SAMtools v1.5, PCR duplicates were filtered with Picard MarkDuplicates, reads overlapping with the hg19 blacklist regions were discarded. The bigWig files with the ratio of RPKM-normalized ChIP-seq signal to the input in 50 bp bins were generated using deepTools2 with ‘--pseudocount’ set to 0 1 and ‘--smoothLength’ of 3 bins. Peaks were called using MACS2 with broad-cutoff of 0.1 (H3K9me3 and H3K27me3) in broad peak mode, qvalue of 0.05 (H3K27ac and H3K4me3) and p-value of 0.001 (CTCF) in narrow peak mode.
Hi-C: Hi-C reads were mapped using bowtie v2.2.3 with the ‘--very-sensitive’ mode and the iterative mapping procedure implemented in hiclib package. Non-uniquely mapped reads, reads mapped on the same fragment and possible PCR duplicates were eliminated. Filtered reads pairs were aggregated into into genomic bins of different sizes to obtain Hi-C contact matrices. Contacts matrices were normalized using the ICE balancing implemented in cooler v0.7.9. Chromatin compartments were annotated using PCA implemented in cooltools v0.3.2 call-compartments function for 100 kb resolution contact matrices. TADs were annotated using the Armatus algorithm implemented in lavaburst package for 20 kb resolution contact matrices.
Genome_build: hg19
Supplementary_files_format_and_content: RNA-seq: normalized signal tracks in bigWig format; read counts per gene in TXT format. ChIP-seq: normalized signal tracks in bigWig format; peaks in BED format. Hi-C: contact matrices in MCOOL format; compartment eigenvectors in TXT format; TADs in BED format.
|
| |
|
| Submission date |
Mar 04, 2021 |
| Last update date |
Nov 22, 2023 |
| Contact name |
Mikhail Magnitov |
| Organization name |
The Netherlands Cancer Institute
|
| Street address |
Plesmanlaan 121
|
| City |
Amsterdam |
| ZIP/Postal code |
1066CX |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL18573 |
| Series (1) |
| GSE168233 |
SETDB1 Fuels the Lung Cancer Phenotype by Modulating Epigenome, 3D Genome Organization, and Chromatin Mechanical Properties |
|
| Relations |
| BioSample |
SAMN18138750 |
| SRA |
SRX10346793 |
