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| Status |
Public on Apr 05, 2021 |
| Title |
SK-LMS-1_H3K27me3 WT ChIP [N1_S10] |
| Sample type |
SRA |
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| Source name |
SK-LMS-1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: SK-LMS-1
cell type: Leiomyosarcoma cells
passages: below 40
genotype/variation: wild type
chip antibody: H3K27me3 (Abcam ab6002)
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| Treatment protocol |
Cells were seeded in 200 mm plate three days before the ChIP. 36h before the ChIP, HDAC4-ER induction was removed (in KO samples) or mantained (in WT samples). At the moment of the ChIP, cross-linking was performed for 15' with 1% formaldeyde (Fisher Scientific, Schwerte, Germany) and then blocked with 125 mM Glycine (Sigma-Aldrich, Darmstadt, Germany).
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| Growth protocol |
SK-LMS-1 cells (HDAC4KO/HDAC4-ER) were grown in DMEM supplemented with 10% FBS, 2mM L-glutamine and penicillin–streptomycin (Euroclone, Milan, Italy). For each ChIP, 3.2*10^6 cells were used.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were obtained with a two-step protocol (nuclei were obtained with an hypotonic buffer (20mM TRIS-HCl pH8; 85 mM KCl; 0.5% NP-40), and nuclear membrane was lysed with Lysis buffer (10mM TRIS pH7.5; 1% NP-40, 0.5% Na-deossicholate; 0.1% SDS); then lysates were clarified from sonicated nuclei (30', medium power, Bioruptor Diagenode) and histone-DNA complexes were isolated with protein A/antibodies: 2μg of anti-H3K27ac (Abcam ab177178) , 3μg of anti-H3K27me3 (Abcam ab6002), 4μg of anti-HDAC4 (Paroni et al, 2004) antibodies or control IgG. Washes were performed in LOW SALT IMMUNE COMPLEX WASH BUFFER (0.1% SDS, 1% TRITON, 20mM Tris-HCl pH8, 150 mM NaCl), HIGH SALT WASH BUFFER (0.1% SDS, 1% TRITON, 2mM EDTA, 20mM Tris-HCl pH8, 500 mM NaCl), LiCl Immune Complex Wash Buffer (0.25M LiCl, 1% NP40, 0.2% Na-deossicolato, 1mM EDTA, 10Mm Tris-HCl pH8) and TE. RNA was digested with RNAseA (2μg). DNA was de-crosslinked and purified with Zymo DNA columns.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, 5 ng of DNA were used. DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
N1_S10
Facultative heterochromatin evaluation in wt cells
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| Data processing |
Basecalls performed using the ShortRead R/Biocoductor package
ChIP-seq reads were aligned to the hg38 genome assembly using bowtie2 version 2.3.4.3 with default configuration for unpaired reads
Duplicate reads were flagged using samtools but no duplicates were removed
Peaks were called using HOMER for HDAC4 ChIP (“factor” mode) and using MACS2 version 2.1.0 for H3K27ac and H3K27me3 (“sharp” mode and “broad” mode, respectively), q<0.05
Genome_build: hg38
Supplementary_files_format_and_content: bigWig files were generated by the deepTools python suite command ‘bamCoverage’. Output files represent the genomic coverage associated with each ChIP-seq experiment, expressed as reads per bin. Bins are short consecutive counting windows of size n=50nt. Data normalized as Reads Per Kilobase per Million mapped reads (RPKM).
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| Submission date |
Apr 30, 2020 |
| Last update date |
Apr 05, 2021 |
| Contact name |
Emiliano Dalla |
| E-mail(s) |
emiliano.dalla@uniud.it
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| Phone |
+390432494286
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| Organization name |
Università degli Studi di Udine
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| Department |
Department of Medicine
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| Street address |
Piazzale Kolbe 4
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| City |
Udine |
| State/province |
UD |
| ZIP/Postal code |
33100 |
| Country |
Italy |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE149644 |
HDAC4 controls senescence and aging by safeguarding the epigenetic identity and ensuring the genomic integrity |
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| Relations |
| BioSample |
SAMN14777731 |
| SRA |
SRX8213856 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4506466_H3K27me3_WT_SKLMS1_coverage.bw |
188.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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