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Sample GSM1004953

Query DataSets for GSM1004953

Status

Public on Sep 20, 2012

Title

SL14522

Sample type

SRA

Source name

Th17 in vitro

Organism

Mus musculus

Characteristics

genotype: Batf +/+
time_point: 48h
application: FAIRE-seq
cell type: Th17

Growth protocol

Naive CD4 T cells were typically cultured under standard non-polarizing Th0 or Th17 differentiation conditions for 48h (or as indicated).

Extracted molecule

genomic DNA

Extraction protocol

RNA was extracted from cells using the Trizol protocol; Transcription factor ChIP/FAIRE-Seq was performed from sonicated, PFA-crosslinked nuclear lysates and for ChIP, TF-DNA complexes were isolated using specific antibodies; Histone ChIP was performed using mono/di-nucleosomes prepared from MNase digested chromatin.
mRNA was prepared from total RNA by poly-A selection and cDNA synthesis was carried out as described (Mortazavi et al., 2008). The resulting dsDNA was prepared for sequencing by ligation of Illumina sequencing adapters, selection of 225 bp fragments from a 2% agarose SizeSelect E-Gel (Invitrogen), and amplification with 15 cycles of PCR using Illumina paired- end primers. Alternatively, some libraries were made using the Nextera tagmentation protocol described (Gertz et al., 2012).

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Data processing

All libraries were sequenced with single-end 36 bp reads on an Illumina GAIIx or single-end 50 bp reads on an Illumina HiSeq 2000
Sequence reads were mapped to the mus musculus genome (version mm9) with Bowtie (version 0.12.7) (Langmead et al., 2009) and with the following settings: -k 1 --best. The --phred33-quals or --phred64-quals parameter was set as needed depending on the format of the input fastq file.
ChIP-seq datasets were further processed to call peaks with the MACS software (version 1.4.0 20110619) using the settings: -p 1e-10 -m 15,30 -s 36 -g mm --bw=200 (Zhang et al., 2008). All were processed against an appropriate control.
RNA-seq datasets were also processed through Tophat (version 1.2.0) with settings: -a 10 -g 20 --no-novel-juncs -G refseqGeneAnnot.gtf (Trapnell et al., 2009).
Tophat results were then pipelined to Cufflinks (version 0.9.3) with the settings: -M 20101217_rRNA_tRNA_mask.gtf -G refseqGeneAnnot.gtf (Trapnell et al., 2010).
Absolute read counts for annotated genomic features were computed using the htseq-count script from the HTSeq (version 0.5.3p3) software suite with parameters: --stranded=no --mode=union.
MACS peaks of multiple TFs were clustered over the genome using a simple R script as described in the accompanying manuscript.
MACS peaks were converted to gene wide scores as decribed in main manuscript
Differential expression analysis for RNA-seq was performed using Deseq with default parameters.
Network inference for RNA-seq compendium was run with the Inferelator with default parameters.
Genome_build: mm9
Supplementary_files_format_and_content: Each ChIP-Seq dataset includes a result file <lib>_<control>_genes.txt, where the <lib> is the ChIP library and <control> its corresponding control library. This is a MACS output file augmented with additional columns generated by the custom R script described in the data processing steps. Each RNA-Seq dataset includes a result file <lib>_genes.expr.txt, which is the output of cufflinks.

Submission date

Sep 17, 2012

Last update date

May 15, 2019

Contact name

Ashish Agarwal

Organization name

Solvuu

Street address

150 E 44th St #27E

City

New York