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Sample GSM3335757

Query DataSets for GSM3335757

Status

Public on Sep 05, 2020

Title

HGPS188 input

Sample type

SRA

Source name

Hutchinson-Gilford progeria syndrome (HGPS) skin fibroblasts

Organism

Homo sapiens

Characteristics

cell type: patient derived skin fibroblast
passage: 12
chip antibody: none

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

Cells were cross-linked with 1% HCHO for 12 minutes at room temperature, lysed and chromatin sheared using Branson Digital Sonifier. IP were performed overnight on a wheel at 4° with 2.4 micrograms of H3K9me3 antibody (ab8898, Abcam). 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) and DNA extracted from beads 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 the automation instrument Biomek FX (Beckman Coulter), 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 at the IEO Genomic Unit in Milan.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

We trimmed reads, using Trimmomatic61 (version 0.32) in single end mode.
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 used the Enriched Domain Detector (EDD) tool to call H3K9me3 peaks, with the following options: --fdr 0.1 –gap-penalty 10 –bin-size 100 –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: H3K9me3 peak coordinates for a specific sample. bigWig: genome wide IP - input signal for a specific sample, smoothed by a Gaussian kernel.

Submission date

Aug 16, 2018

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