|
| Status |
Public on Aug 25, 2010 |
| Title |
mm8-Bcell-KO-dex-H3K4me1 |
| Sample type |
SRA |
| |
|
| Source name |
mouse B cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: Cd19-cre X Paxip flox/flox
age: 6-14 weeks
tissue: spleen
cell type: B cells
genome/variation: PTIP-knockout
antibody: H3K4me1
antibody vendor, cat#: Abcam, ab8895
treatment group: dextran
|
| Treatment protocol |
B cells were stimulated with LPS (25 μg/ml; Sigma), and either α-IgD-dextran (2.5ng/ml; FinaBio) or IL4 (5 ng/ml; Sigma) for 2 days as indicated.
|
| Growth protocol |
B cells were isolated from spleens of 6- to 14-week-old mice by immunomagnetic depletion with α-CD43 beads (Miltenyi Biotech).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Resting or 2 day stimulated B cells were harvested for chromatin preparation, and ChIP and Illumina sequencing was performed. Briefly, for histone modification ChIPs, cells were washed and digested with MNase to generate native mononuclesomal chromatin fragments. For Pol II and PTIP ChIPs, cells were treated with 1% formaldehyde for 10min at room temperature to generate cross-linked chromatin. Chromatin from 10^7 cells was used for each ChIP experiment. DNA was quantified using PicoGreen (Invitrogen) prior to library preparation. Libraries were prepared by making blut-end DNA with End-It end repair kit (Epicentre #ER81050) according the manufacturer's instructions. The blunt, phosphorylated ends were treated with Taq DNA polymerase (5U/ul, Invitrogen #18038-042) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, DNA was PCR amplified with Illumina primers for 18 cycles and library fragments of ~220 bp were band isolated from an agarose gel. The purified DNA quantified using PicoGreen (Invitrogen) and was captured on an Illumina flow cell for cluster generation using Single-Read Cluster Generation Kit v2. Libraries were sequenced on the Genome Analyzer following the SBS Sequencing 36 Cycle Kit v3 protocol.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Chromatin IP
All experiments were performed with mice generated by crossing Cd19-cre mice (as described in Rickert et al. B lymphocyte-specific, Cre-mediated mutagenesis in mice. Nucleic Acids Res. 1997 Mar 15;25(6):1317-8.) with Paxip flox/flox mice (as described in Kim et al. Pax transactivation-domain interacting protein is required for urine concentration and osmotolerance in collecting duct epithelia. J Am Soc Nephrol. 2007 May;18(5):1458-65.).
|
| Data processing |
Sequence reads were obtained and aligned to the mouse genome (mm8) using the standard Illumina Genome Analyzer Pipeline. Only uniquely mapped reads were retained. BedGraph data files were built by summing sequence read numbers into 200bp (or 400bp for PolII) non-overlapping windows, with read position shifted 75bp (or 150bp for PolII) to represent the DNA fragment center. More details as described in Daniel et al., 2010.
|
| |
|
| Submission date |
Mar 12, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Chongzhi Zang |
| Organization name |
University of Virginia
|
| Street address |
P. O. Box 800717
|
| City |
Charlottesville |
| State/province |
VA |
| ZIP/Postal code |
22908 |
| Country |
USA |
| |
|
| Platform ID |
GPL9185 |
| Series (1) |
| GSE20852 |
PTIP promotes chromatin changes critical for immunoglobulin class switch recombination |
|
| Relations |
| SRA |
SRX018151 |
| BioSample |
SAMN00010564 |
