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Sample GSM4674924

Query DataSets for GSM4674924

Status

Public on Sep 05, 2020

Title

CTRL004 SAMMY-seq S3, late passage replicate 1

Sample type

SRA

Source name

CTRL004 SAMMY-seq S3, late passage

Organism

Homo sapiens

Characteristics

cell type: patient derived skin fibroblast
fraction: S3
passage: 27

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

4 million 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 3000 rpm 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 25U of RNase–free DNase (Invitrogen) in CSK buffer for 30 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 5000 rpm 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 5000 rpm 3 min at 4°C and the supernatant was labeled as S3 fraction. This treatment removed the majority of histones from chromatin. After 2 washing 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.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

processed data (*S4vsS3) files are linked to the S4 sample records

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 = 0 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 45 --bin-size 200 --write-log-ratios --write-bin-scores and also excluding blacklisted genomic regions containing telomeric, centromeric, and certain heterochromatic regions62. We also changed the required_fraction_of_informative_bins parameter to 0.98.
Genome_build: hg38-noalt
Supplementary_files_format_and_content: bed: 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

Jul 16, 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