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Sample GSM1668939

Query DataSets for GSM1668939

Status

Public on Dec 04, 2015

Title

CB4_Input_RK063

Sample type

SRA

Source name

germinal center B cells from tonsil tissues

Organism

Homo sapiens

Characteristics

tissue: Germinal center B cells
antibody: no antibody

Treatment protocol

Purified GC B cells were cross-linked with 1% formaldehyde for 10 min at room temperature, quenched by the addition of glycine to a final concentration of 0.125 M, and frozen.

Growth protocol

Germinal center B cells were isolated from human tonsil tissue by magnetic cell separation (CD77+), as described in Klein et al, Proc Natl Acad Sci U S A, 2003.

Extracted molecule

genomic DNA

Extraction protocol

Cell lysis and nuclei isolation was performed using the TruChIP Chromatin Shearing Kit High Cell SDS (Covaris). Nuclei were sonicated using the S220 Ultrasonicator (Covaris) in order to obtain chromatin fragments of 200-500bp. ChIP was performed using anti-BCL6 (N3, Santa Cruz) or the anti-FOXO1 (Abcam, ab39670) antibodies. ChIP DNA was isolated by the MinElute DNA isolation kit (Qiagen).
ChIP-seq libraries were constructed starting from 4ug of ChIP or Input DNA as reported in Blecher-Gonen et al., 2013, Nature protocol. Libraries were quantified using the KAPA SYBR FAST Universal qPCR Kit (KAPA Biosystems), normalized to 10nM, pooled and sequenced in an Illumina HiSeq 2500 instrument as single-end 100 bp reads, obtaining on average 25x10^6 reads/sample.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Data processing

Sequencing data were acquired through the default Illumina pipeline using Casava V1.8.
Reads were aligned to the human genome (UCSC hg19) for human GC B cells using the Bowtie2 aligner v2.1.0, allowing up to two mismatches. Duplicate reads (i.e. reads of identical length mapping to exactly the same genomic locations) were removed with samtools v0.1.19 using the rmdup option.
Reads were normalized to total reads aligned (counts per million). Peak detection was done using ChIPseeqer v2.0, enforcing a minimum fold change of 2 between ChIP and input reads, a minimum peak width of 100 bp, and a minimum distance of 100 bp between peaks.
The threshold for statistical significance of peaks was set at 10^-5 for the FOXO1 samples and 10^-12 for the BCL6 samples.
Genome_build: hg19
Supplementary_files_format_and_content: bedgraph files contain peak locations. Columns 1, 2, and 3 contain the chromosome, start, and end position of the peak respectively. The fourth column is the maximum peak height normalized per million mapped reads in each library.

Submission date

Apr 28, 2015

Last update date

May 15, 2019

Contact name

Riccardo Dalla-Favera

E-mail(s)

rd10@cumc.columbia.edu

Organization name

Columbia University

Street address

1130 St Nicholas Avenue