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Sample GSM494702

Query DataSets for GSM494702

Status

Public on Jan 08, 2010

Title

IgG bound post IL21 in B cells

Sample type

SRA

Source name

B cells

Organism

Mus musculus

Characteristics

genotype/variation: wildtype
antibody: IgG
stimulation: post IL21

Treatment protocol

CD4+ T cells were treated with IL-21 (100ng/ml) for 1 hour.

Growth protocol

CD4+ T cells were isolated from mouse spleen using anti-CD4-magnetic beads (Miltenyi). For pre-activation of CD4+ T cells, cells were stimulated with 3 μg/ml plate-bound anti-CD3 and 1 μg/ml soluble anti-CD28 for 3 days, and rested for 24 h in fresh RPMI medium.

Extracted molecule

genomic DNA

Extraction protocol

Chromatin from approximately 2x10^7 cells were used for each ChIP experiment. End repair for ChIP fragments was performed using PNK and Klenow enzyme, followed by treatment with Taq polymerase. Sequencing adaptors were ligated to the repaired ends and ChIP DNA was amplified using adaptor primers for 17 cycles. Fragments around 200bp were isolated from agarose gel and purified DNA was used directly for cluster generation and sequencing following the manufacturer protocols.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer II

Description

Chromatin IP against IgG

Data processing

To align sequence tags, analyze their clustering, identify binding motifs, and display results, new code was developed in C language as extensions of the AceView project (Thierry-Mieg and Thierry-Mieg, 2006). Each Solexa 25-mer tag was mapped on the mouse reference genome (version 37), allowing up to 2 mismatches (single base insertion, deletion, or substitution), and retained only if it had a single best position genome-wide. Each tag was shifted downstream by half the effective length of the sonicated sequenced fragments, measured as the maximum of the genome-wide correlation function between hits to the sense and antisense strands of the chromosomes, and represented by an elementary Gaussian with an area of 1 and s of 100 bp, which dampens the sampling fluctuations and introduces a controlled level of fuzziness and “smoothness”. Summing these elementary Gaussians yields a smoothed tag density. Regions where the density locally exceeds a given threshold are analyzed. The number of tags was measured after subtracting the local tags from the matched IgG experiment. If the tag number exceeded a second threshold, it was scored as a peak. Regions with an IgG peak were considered non-specific and eliminated. The peak height indicates the strength of protein binding. The position of the maximum of the peak (i.e., the maximum of the probability of binding the protein) is expected to coincide with the binding motif. The width covered by the peak (typically 200-600 bp) is the region protected by the protein or protein complex. Often, in large peaks, one can distinguish sub-peaks that are expected to correspond to the binding of multiple proteins. When multiple proteins bind to overlapping sequences, their peaks are clustered to generate a broader composite binding site.

Submission date

Jan 08, 2010

Last update date

May 15, 2019

Contact name

Dustin E Schones

E-mail(s)

dschones@coh.org

Organization name

City of Hope

Street address

1500 E Duarte Rd.