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| Status |
Public on Dec 04, 2015 |
| Title |
CB4_BCL6_RK040 |
| Sample type |
SRA |
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| Source name |
germinal center B cells from tonsil tissues
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Germinal center B cells
antibody: anti-BCL6, N3 (Santa Cruz)
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| 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.
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| 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.
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| 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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| 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.
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| Submission date |
Apr 28, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Riccardo Dalla-Favera |
| E-mail(s) |
rd10@cumc.columbia.edu
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| Organization name |
Columbia University
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| Street address |
1130 St Nicholas Avenue
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE68349 |
ChIPseq analyses in germinal center B cells |
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| Relations |
| BioSample |
SAMN03570600 |
| SRA |
SRX1012538 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1668937_Peaks.CB4_BCL6_RK040_vs_Input_RK063_p12.bedgraph.gz |
121.9 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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