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Sample GSM4595508

Query DataSets for GSM4595508

Status

Public on Sep 05, 2020

Title

CTRL004 S2 fraction 250K rep2

Sample type

SRA

Source name

CTRL004 S2 fraction 250K

Organism

Homo sapiens

Characteristics

cell type: patient derived skin fibroblast
passage: 18
fraction: S2

Growth protocol

Primary fibroblast cell lines were cultured in DMEM High glucose with glutamax supplemented with 15% FBS and 1% Pen/Strep.

Extracted molecule

genomic DNA

Extraction protocol

250 thousand, 50 thousand or 10 thousand fibroblasts were washed in PBS 1X, and extracted in cytoskeleton buffer (CSK: 10 mM PIPES pH 6,8; 100 mM NaCl; 1 mM EGTA; 300 mM Sucrose; 3 mM MgCl2; 1X protease Inhibitors by Roche Diagnostics; 1 mM PMSF) supplemented with 1 mM DTT and 0,5% Triton X-100. After 5 min at 4°C the cytoskeletal structure was separated from soluble proteins by centrifugation at 845 g for 3 min, and the supernatant was labeled as S1 fraction. The pellets were washed with an additional volume of cytoskeleton buffer. Chromatin was solubilized by DNA digestion with 8U of RNase–free DNase (Invitrogen) in CSK buffer for 60 min at 37°C. To stop digestion, ammonium sulphate was added in CSK buffer to a final concentration of 250 mM and, after 5 min at 4°C samples were pelleted at 2350 g for 3 min at 4°C and the supernatant was labeled as S2 fraction. After a wash in CSK buffer, the pellet was further extracted with 2M NaCl in CSK buffer for 5 min at 4°C, centrifuged at 2350 g 3 min at 4°C and the supernatant was labeled as S3 fraction. This treatment removed the majority of histones from chromatin. After 2 washings in NaCl 2M CSK, the pellets were solubilized in 8M urea buffer to remove any remaining protein component by applying highly denaturing conditions. This remaining fraction was labeled as S4.
We added to each fraction TE buffer to reach a final volume of 200μL. After incubation with RNAse cocktail (Ambion) (90 minutes at 37°) and Proteinase K (Thermo Scientific) (150 minutes at 55°), DNA was extracted by standard phenol/chloroform extraction, precipitated and resuspended in 16μl of nuclease-free water. Then S3 and S4 samples were transferred to microTUBE-15 AFA Beads Screw-Cap (Covaris) and sonicated with Covaris M220 focused-ultrasonicator (water bath 20°C, peak power 30.0, duty factor 20.0, cycles/burst 50, duration 150 seconds). S2 samples were further purified using PCR DNA Purification Kit (Qiagen) and suspend in 30μL of nuclease-free water and they were not sonicated. All the samples were quantified using Qubit HS DNA kit (Life Technologies) and library prepared with NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) using Unique Dual Index NEBNext Multiplex Oligos for Illumina (NEB). Library were quantified using Qubit HS DNA kit (Life Technologies) and profiles were checked by capillary electrophoresis on a Agilent 2100 Bioanalyzer. Finally, DNA libraries were sequenced using an Illumina NovaSeq 6000 instrument according to manufacturer’s instructions.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NovaSeq 6000

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.

Submission date

Jun 08, 2020

Last update date

Sep 05, 2020

Contact name

Chiara Lanzuolo

E-mail(s)

[email protected]

Phone

0200660358

Organization name

CNR and Istituto Nazionale Genetica Molecolare