|
| Status |
Public on Mar 01, 2013 |
| Title |
RPA_ChIPSeq_Rif1_panel4B |
| Sample type |
SRA |
| |
|
| Source name |
Primary B cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Splenocyte (B lymphocytes)
genotype: Rif1F/FCd19Cre/+
chip antibody: anti-RPA32 Calbiochem NA19L, lot#D00127062
|
| Growth protocol |
CD43- resting B cells were isolated from mouse spleens by negative selection with magnetic beads (Miltenyi Biotech). Following isolation, resting B cells were stimulated with LPS (50 mcg/ml), IL-4 (2.5 ng/ml) and anti-Cd180 (RP105) (250 ng/ml) for 72 hrs.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% paraformaldehyde at 37°C for 10 minutes followed by quenching with glycine and sonication. Chromatin fragments were then immunoprecipitated with the antibodies shown in the sample characteristics following standard protocols. Immunoprecipitates were processed following Illumina’s protocol and sequenced on a HiSeq 2000.
The ChIP-DNA was blunt-ended with End-It DNA end repair kit (Epicentre) and A-tailed with Taq DNA polymerase (Invitrogen) in the presence of 200mM of dATP for 40 minutes at 70°C. The sample was purified by phenol-chloroform extraction after each reaction. Illumina compatible adaptors (Illumina or Bioo Scientific) were then ligated with T4 DNA ligase (Enzymatics) and the reaction was purified once with AMpure XP magnetic beads (Beckman Coulter). Samples were PCR amplified for 18-cycles with KAPA HiFi DNA polymerase mix (KAPA biosystems). The amplicon was run on a 2% agarose gel and size-selected at 200-300 bp.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
Basecalls performed using CASAVA version 1.4
ChIP-seq reads were aligned to the mm9 genome assembly using bowtie requiring unique alignment (-m 1)
Uniquely aligned reads were analyzed by SICER using an expectation value E of 0.05 in a random background model
Genome_build: mm9
Supplementary_files_format_and_content: wig files were generated using SICER
|
| |
|
| Submission date |
Nov 14, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Thiago Y Oliveira |
| E-mail(s) |
toliveira@rockefeller.edu
|
| Organization name |
The Rockefeller University
|
| Department |
Immunology, Virology and Microbiology
|
| Lab |
Laboratory of Molecular Immunology
|
| Street address |
1230 YORK AVE
|
| City |
NEW YORK |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE42298 |
Rif1 Prevents Resection of DNA Breaks and Promotes Immunoglobulin Class Switching |
|
| Relations |
| SRA |
SRX204595 |
| BioSample |
SAMN01814404 |
| Named Annotation |
GSM1037392_r120605A_l1i2_ab180_Rif1flfl_Cd19Cre_RPA_repb.wig.gz |
| Named Annotation |
GSM1037392_r120605A_l1i2_ab180_Rif1flfl_Cd19Cre_RPA_repb_by_strand_500.wig.gz |
