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Sample GSM2902658

Query DataSets for GSM2902658

Status

Public on Dec 01, 2023

Title

HDF_TGFb1_YAP_Rep1

Sample type

SRA

Source name

HDF

Organism

Homo sapiens

Characteristics

cell type: Dermal fibroblast (LONZA CC-2511)
passage: Passage 9
treatment: 5 ng TGFb1/mL for 24h
chip antibody: anti-YAP: ab52771 (Abcam), Research Resource Identifier: AB_2219141

Treatment protocol

Starvation and experiments were performed in DMEM containing 50 U/L penicillin/streptomycin, 0.5% FBS and 17 mM ascorbic acid (A8960; Sigma-Aldrich). Human dermal fibroblasts (15,000/cm2) were serum starved for 16 hours and subsequently exposed to TGFβ1 for 24 hours.

Growth protocol

Primary human dermal fibroblasts (CC-2511; Lonza, Basel, Switzerland) were sub-cultured in Dulbecco’s modified Eagle medium (DMEM, 12-604F; Lonza) containing 50 U/L penicillin/streptomycin (15140122; Thermo Scientific) and 10% fetal bovine serum (FBS; Sigma-Aldrich). Before commencing experiments, cells were verified to be mycoplasm free.

Extracted molecule

genomic DNA

Extraction protocol

Protein complexes were cross-linked with 1.5 mM ethylene glycol bis(succinimidyl succinate) (21565; Thermo Scientific) in PBS with gentle agitation at RT for 15 minutes. Cross-linking of protein complexes to DNA was performed by adding methanol-free formaldehyde to a final concentration of 1% and incubation with gentle agitation at RT for 8 minutes. Next, cross-linking was quenched by adding glycine to a final concentration of 200 mM at RT for 5 minutes. Cells were then washed three times with ice-cold PBS containing 1% protease inhibitor cocktail, collected by scraping, and pelleted by centrifugation at 2000 × g for 5 minutes at 4°C. Nuclei were purified by re-suspending pellets in buffer LB1 (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1% protease inhibitor cocktail) and washed in buffer LB2 (10 mM Tris HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% protease inhibitor cocktail) followed by lysis in buffer LB3 (10 mM Tris HCl p.H 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine, 1% NP-40, 1% Triton X-100, 1% protease inhibitor cocktail) while rotating for 1 hour at 4°C. Chromatin samples were sheared twice using a BioRuptor PLUS device (B01020001; Diagenode, Liège, Belgium) for 6 minutes at 4°C. Correct chromatin fragment size was verified using gel electrophoresis. Dynabeads (10002D; Thermo Fisher Scientific) were incubated for 4 hours with primary antibodies. YAP bound chromatin was precipitated in RIPA buffer (50 mM HEPES pH 7.6, 1 mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5 M LiCL, 1% protease inhibitor cocktail) at 4°C on a rotating platform for 18 hours. Afterwards the beads were washed five times in RIPA buffer, and once with TE buffer (10 mM Tris, 1mM EDTA, pH 8.0). Immunoprecipitated chromatin was eluted with elution buffer (1% SDS, 0.1M NaHCO3), de-crosslinked overnight, and cleaned up using RNAse and Protease-K treatment, followed by purification with a PCR cleanup kit (Qiagen).
Sequencing libraries were prepared for input control and two biological replicates for YAP in each culture condition using the NEBNext Ultra II DNA Library Prep Kit according to manufacturer's instructions (E7370; New England Biolabs, USA). Library quality was verified using BioAnalzyer.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

NextSeq 550

Data processing

For Chip-seq, reads were aligned to the human genome build hg19 using BWA (v0.7.12) with default settings. PCR duplicates and unmapped reads were removed with SAMTools (v0.1.19).
Normalized peak coverage files (bigwig) as reads per genomic content, were generated with DeepTools bamCoverage (2.5.1) with 10bp bins from merged (SamTools) with the following parameters: --normalizeTo1x 2451960000 --extendReads 200 -bs 10 --ignoreDuplicates --bl. Blacklisted regions (downloaded from ENCODE) were excluded.
Genome_build: hg19
Supplementary_files_format_and_content: bigwig from merged YAP Chip-seq sample replicates and input DNA

Submission date

Dec 26, 2017

Last update date

Dec 01, 2023

Contact name

Bram Piersma

E-mail(s)

[email protected]

Organization name

University Medical Center Groningen

Department

Pathology and Medical Biology