|
| Status |
Public on Mar 03, 2013 |
| Title |
GCB_BCL6_ChIP |
| Sample type |
SRA |
| |
|
| Source name |
Primary cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Germinal center B cells
chip antibody: BCL6
|
| Growth protocol |
Germinal center B-cells were isolated from fresh deidentified human tonsils derived from surgery waste with the approval of the Human Research Protections Programs, Division of Research Integrity of the Weill Cornell Medical College in accordance with the Declaration of Helsinki. Purifications were based on the expression of CD77 (GC). Separation was performed using a magnetic beads cell separation system with double positive selection settings (MACS, Miltenyi Biotec). Purity of the isolated cells was confirmed by FACS and always exceeded 95%.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq libraries were prepared using the Illumina ChIP-seq Library preparation Kit following the manufacture's instructions with minor modifications. Briefly 10ng of purified ChIP DNA was end repaired by conversion of overhangs to phosphorylated blunt ends. A' bases were added to the 3'ends of the DNA fragments and Illumina adapters (1:30 dilution) were ligated to the ends of ChIP fragments. After adapter ligation DNA was separated by electrophoresis and size selected by isolating a gel band of 250±25bp. This fragment range corresponds to a ChIP fragment range of about 158±25bp. Size selected fragments were PCR amplified for 15cycles using Illumina genomic DNA primers 1.1 and 1.2 with the following program (30s at 98oC, 15cycles of 10 at 98oC, 30s at 65oC,30s at 72oC and 5min extension at 75oC). Libraries were quantified and validated using Agilent Technologies 2100 Bioanalyser for size, concentration and purity. Q-PCR was repeated to confirm retention of relative enrichment.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
ChIP-seq in GC B cells against BCL6
|
| Data processing |
Raw images generated went through primary image analysis and basecalling (RTA v1.6) that was followed by Illumina Genome Analyzer Off-Line Basecaller (OLB v1.6) analysis, where reads were aligned to the human genome (UCSC hg18) using ELAND. Only sequences mapped uniquely to the genome with not more than 2 mismatches were used for downstream analysis. Several reads mapping to the same exact location (clonal reads) were considered amplification artifact and were excluded from the analysis. Read density tracks were generated using ChIPseeqer and visualized using the UCSC browser.
Genome_build: hg18
Supplementary_files_format_and_content: "Read_density-wig" files are wig tracks generated using ChIPseeqerMakeReadDensityTrack of the ChIPseqer package. (http://icb.med.cornell.edu/wiki/index.php/Elementolab/ChIPseeqerMakeReadDensityTrack).They represent the average ChIPseq read density, normalized to the total number of reads.
|
| |
|
| Submission date |
Mar 20, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Katerina Hatzi |
| E-mail(s) |
kac2029@med.cornell.edu
|
| Organization name |
WCMC
|
| Department |
Hematology/Oncology
|
| Lab |
Ari Melnick
|
| Street address |
413 E 69th Street, BB-1462
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10021 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE43350 |
Lineage-specific functions of Bcl6 in immunity and inflammation are mediated through distinct biochemical mechanisms |
|
| Relations |
| SRA |
SRX130608 |
| BioSample |
SAMN00829330 |
