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| Status |
Public on Sep 05, 2020 |
| Title |
HGPS167 H3K27me3 ChIP-seq |
| Sample type |
SRA |
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| Source name |
HGPS167 H3K27me3 ChIP-seq
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| Organism |
Homo sapiens |
| Characteristics |
cell type: patient derived skin fibroblast
passage: 12
chip antibody: 07-449, Millipore
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| Growth protocol |
Primary fibroblast cell lines were cultured in DMEM High glucose with glutamax supplemented with 15% FBS and 1% Pen/Strep.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross-linked with 1% HCHO for 12 minutes at room temperature, lysed and chromatin sheared using Covaris M220 focused-ultrasonicator. IP were performed overnight on a wheel at 4° with 2 micrograms of H3K27me3 antibody (07-449, Millipore). The following day, antibody-chromatin immunocomplexes were loaded onto Dynal G magnetic beads (Invitrogen 10004D); the bound complexes were washed once in Low Salt Solution (0,1% SDS, 2mM EDTA, 1% Triton X-100, 20mM Tris pH 8, 150 mM NaCl), once in High Salt Solution (0,1% SDS, 2mM EDTA, 1% Triton X-100, 20mM Tris pH 8, 500 mM NaCl), once again in Low Salt Solution and once in Tris/EDTA 50 mM NaCl. Crosslinking was reversed at 65°C overnight in Elution Buffer (50 mM Tris pH 8, 10 mM EDTA, 1% SDS). DNA purified by standard phenol/chloroform extraction, precipitated and resuspended in 30 μl of 10 mM Tris pH 8. ChIP efficiency was tested by qPCR reactions, performed in triplicate (precipitated DNA samples as well as input DNA) using QuantiTect SYBR Green master mix (Qiagen) on a StepOnePlus™ Real-Time PCR System (Applied Biosystems). Relative enrichment was calculated as ChIP / Input ratio.
Libraries for sequencing were created using NEBNext Ultra II DNA Library Prep Kit (NEB), then qualitatively and quantitatively checked using Agilent High Sensitivity DNA Kit (Agilent Technologies, 5067-4627) on a Bioanalyzer 2100 (Agilent Technologies). Libraries with distinct adapter indexes were multiplexed and, after cluster generation on FlowCell, were sequenced for 50bp in the single read mode on a HiSeq 2000 sequencer (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Reads were aligned to the hg38-noalt reference human genome available in the bcbio-nextgen pipeline, using bwa aln (version 0.7.12) with options -n 2 -k 2 and saved the results in sam format with bwa samse.
The sam files were converted to bam and name sorted with samtools (version 1.3.1). We marked PCR duplicates using the biobambam2 toolset (version 2.0.54). We discarded reads mapping to non-autosomal chromosomes, PCR duplicates, qcfail, multimapping and unmapped reads with samtools.
We calculated the genome wide IP - input signal for all samples, using the SPP package (version 1.15.4). We imported bam files into the R (version 3.3.1) statistical environment, and selected informative reads with the get.binding.characteristics and select.informative.tags functions, removed anomalous positions with extremely high number of reads using the remove.local.tag.anomalies function, and calculated the differential signal, smoothed by a Gaussian kernel, using the get.smoothed.tag.density function with the default bandwidth and tag.shift parameters.
We called H3K27me3 ChIP-seq peaks with the get.broad.enrichment.clusters function of SPP on the preprocessed data with window.size = 2000 and z.thr = 3, tag.shift = ((cross correlation peak position) / 2) parameters.
We used the Enriched Domain Detector (EDD) tool to select significantly enriched SAMMY-seq domains by comparing less accessible fractions to more accessible ones (S3 vs S2, S4 vs S3 and S4 vs S2 comparisons) in each sample, with the following options: --gap-penalty 25 --bin-size 250 --write-log-ratios --write-bin-scores and also excluding blacklisted genomic regions containing telomeric, centromeric, and certain heterochromatic regions. We also changed the required_fraction_of_informative_bins parameter to 0.98.
Genome_build: hg38-noalt
Supplementary_files_format_and_content: bed: H3K27me3 peak coordinates or SAMMY-seq enriched domain coordinates for a specific sample. bigWig: genome wide IP - input signal for a specific sample, smoothed by a Gaussian kernel.
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| Submission date |
Jun 08, 2020 |
| Last update date |
Sep 05, 2020 |
| Contact name |
Chiara Lanzuolo |
| E-mail(s) |
[email protected]
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| Phone |
0200660358
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| Organization name |
CNR and Istituto Nazionale Genetica Molecolare
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| Lab |
Lanzuolo Lab
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| Street address |
Via Francesco Sforza 35
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| City |
Milan |
| State/province |
--- |
| ZIP/Postal code |
20122 |
| Country |
Italy |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE118633 |
SAMMY-seq, H3K9me3 and H3K27me3 ChIP-seq and RNA-seq of control and progeria fibroblasts |
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| Relations |
| BioSample |
SAMN09727152 |
| SRA |
SRX8362134 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4595499_HGPS167_H3K27me3-spp-gb.bigwig |
505.5 Mb |
(ftp)(http) |
BIGWIG |
| GSM4595499_HGPS167_H3K27me3-spp.broadpeak.gz |
1.8 Mb |
(ftp)(http) |
BROADPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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