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Sample GSM4970358

Query DataSets for GSM4970358

Status

Public on Apr 15, 2021

Title

RNAseq_HEK293-GR_siNON_dex_rep2

Sample type

SRA

Source name

HEK293-GR

Organism

Homo sapiens

Characteristics

cell line: HEK293-GR
medium: steroid-depleted medium, 48h
treatment: dexamethasone, 100nM, 6h
transfection: siNON, 20nM, 72h

Treatment protocol

ChIP-seq: Cells were treated with vehicle (ethanol, EtOH) or dex (100 nM) for 1 h before harvesting. Then cells were fixed with 1% (v/v) formaldehyde for 10 min at room temperature. Chromatin was fragmented to ∼200–400 bp using sonication (Bioruptor UCD-300, Diagenode). Anti-BCOR antibodies (Bethyl Laboratories, A301-673A) were coupled to protein-A- beads (Millipore), fragmented chromatin was incubated with antibody-coupled beads overnight, washed, eluted and de-crosslinked in the presence of proteinase K (Fermentas).
RNA-seq: Cells were transfected with 20 nM ON-TARGETplus SMARTpool siRNAs (Dharmacon) against BCOR (siBCOR; L-004584–01-0005) for 72 h using Lipofectamine RNAiMAX (Invitrogen) reagent and exposed to 100 nM dex or equal volume of vehicle (ethanol, EtOH) for 6 h before collecting. ON-TARGETplus Non-Targeting Pool (GE Dharmacon, Lafayette, LA) was used as the control siRNA (siNON).

Growth protocol

ChIP-seq: HEK293-GR cells were grown in steroid-depleted medium (DMEM supplemented with 2.5% (v/v) charcoal-treated FBS) for 2 days.
RNA-seq: HEK293-GR cells were grown on 6-well plates (300 000 cells/well) in regular growth medium for 24h after which the medium was replaced with steroid-depleted medium (DMEM supplemented with 2.5% (v/v) charcoal-treated FBS).

Extracted molecule

polyA RNA

Extraction protocol

ChIP-seq: Chromatin fragments were purified using MiniElute columns (Qiagen), ChIP-seq libraries were prepared using NEBNext kit (New England Biolabs) and sequenced with HiSeq2000 at EMBL GeneCore (Heidelberg, Germany).
RNA-seq: Total RNA was extracted from four biological replicates using TriPure isolation reagent (Roche) and treated with DNAse. RNA-seq libraries were prepared using NEBNext Poly(A) mRNA Magnetic isolation module (New England Biolabs) and sequenced with Illumina HiSeq2000 at EMBL GeneCore (Heidelberg, Germany).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

RNA-seq from siNON and dexamethasone-treated cells.
RNAseq-DEseq2_HEK293-GR_siNON-vs-siBCOR_EtOH-vs-dex.txt

Data processing

For ChIP-seq, The quality of raw reads was analyzed by FastQC and FASTX-toolkit was used for preprocessing raw reads (removal of artefacts, trimming reads, quality filtering and collapsing identical reads). Preprocessed reads were aligned against human genome hg19 with Bowtie software (version 0.12.9) with the command line: -v 1 -k 1 -m 1 -f -S --best hg19. Peak calling was done in HOMER (version 4.10.3) using findPeaks command (with parameters: -F 3, -tagTreshold 20, -minDist 350) and appropriate input as a control. Two replicates were compared with each other using bedTools (intersectBed) and only those binding sites found in both replicates were considered representative and used for further analysis. Representative BCOR binding sites from vehicle and dexamethasone samples were merged and sites with two-fold difference in RPKM-normalized ChIP-seq signal between vehicle- or dexamethasone-treatments were defined vehicle-enriched (V-enriched), dexamethasone-enriched (D-enriched), and shared (difference less than two-fold between vehicle and dexamethasone samples).
For RNA-seq, the quality of raw reads was analyzed by FastQC and trimmomatic (LEADING:5, TRAILING:5, MINLEN:36) and HOMER (homerTools trim -3 AAAAAAAA -mis 1 -min 36) were used for preprocessing raw reads (removal of artefacts, trimming reads, removing poly-A). Remaining reads were mapped to reference human transcriptome and genome (hg19) using tophat2. Table of transcript-level counts from exon regions was generated using HOMER (analyzeRepeats.pl rna hg19 -noadj -count exons).
Genome_build: hg19
Supplementary_files_format_and_content: ChIP-seq files of V-enriched, D-enriched and shared BCOR chromatin binding sites are in bed format. A text file with table of transcript-level counts is provided for RNA-seq data.

Submission date

Dec 10, 2020

Last update date

Apr 15, 2021

Contact name

Einari A Niskanen

E-mail(s)

[email protected]

Organization name

University of Eastern Finland

Department

School of Medicine