|
| Status |
Public on Jun 13, 2023 |
| Title |
priB_Rif1FH_ChIPseq_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
priB
|
| Organism |
Mus musculus |
| Characteristics |
cell type: primary B cells
genotype: Rif1FH/FH
antibody: anti-CD43 MicroBeads
|
| Growth protocol |
primary B cells were isolated from Rif1FH/FH and C57BL/6 (Rif1WT/WT) mice spleens using anti-CD43 MicroBeads (Miltenyi Biotec) and expanded in complete RPMI containing 5 µg/ml LPS (Sigma-Aldrich) and 5 ng/ml mouse recombinant IL-4 (Sigma-Aldrich) to allow B cell activation and class switch recombination to IgG1. B cells were harvested 72h after activation and 4 x 10^7 cells
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation was performed as described Foti et al. 2021. In summary, primary B cells were isolated from Rif1FH/FH and C57BL/6 (Rif1WT/WT) mice spleens using anti-CD43 MicroBeads (Miltenyi Biotec) and expanded in complete RPMI containing 5 µg/ml LPS (Sigma-Aldrich) and 5 ng/ml mouse recombinant IL-4 (Sigma-Aldrich) to allow B cell activation and class switch recombination to IgG1. B cells were harvested 72h after activation and 4 x 107 cells were cross-linked by using 2 mM disuccinimidyl glutarate (ThermoFisher 20593) in PBS for 45 min and 1% formaldehyde for 5 min (Thermo Scientific 28908). The reaction was quenched with 0.125 M glycine. Cells were washed thrice with ice-cold PBS and lysed in SDS lysis buffer. Chromatin fragmentation was performed using a Covaris E220 sonicator to obtain fragments between 200 and 600 bp. Chromatin was quantified with a ND-1000 NanoDrop spectrophotometer. Immunoprecipitation was performed with 0.5 μg of anti-HA antibody (Santa Cruz sc-7392) and 50 µl of Dynabeads protein G (Thermofisher 10003D) and 25 μg chromatin
ChIP libraries were prepared by NEBNext ultra II DNA library preparation kit (NEB E7645L) and sequenced on one lane of a NovaSeq 6000 (Illumina) machine
4 x 10^7 cells were cross-linked by using 2 mM disuccinimidyl glutarate in PBS for 45 min and 1% formaldehyde for 5 min . The reaction was quenched with 0.125 M glycine. Cells were washed thrice with ice-cold PBS and lysed in SDS lysis buffer. Chromatin fragmentation was performed using a Covaris E220 sonicator to obtain fragments between 200 and 600 bp. Chromatin was quantified with a ND-1000 NanoDrop spectrophotometer. Immunoprecipitation was performed with 0.5 μg of anti-HA antibody (Santa Cruz sc-7392) and 50 µl of Dynabeads protein G (Thermofisher 10003D) and 25 μg chromatin
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
ChIP-seq
Raw reads were trimmed with trim_galore v0.6.4 (https://github.com/FelixKrueger/TrimGalore) setting minimum length to 18 bases and default arguments otherwise
The trimmed reads were aligned to aligned to the mm9 reference genome with bowtie v1.0.0 with -S --trim5 0 --trim3 0 -v 2 --best --strata --tryhard -m 1 --phred33-quals --chunkmbs 256
Genome coverage tracks were computed with deeptools bamCoverage v3.3.0 using command-line parameters --normalizeUsing CPM --exactScaling –binSize 50 and –ignoreDuplicates
Log2 ratio tracks were computed with deeptools bamCompare v3.3.0 using command-line parameters --scaleFactorsMethod readCount --operation log2 --pseudocount 1 –binSize 50 –ignoreDuplicates.
RIF1 narrow and broad peaks were called for each replicate with MACS v2.2.6, RIF1 associated domains (RADs) were called using EDD v1.1.19. RADs, narrow and broad peaks and domains were merged using bedtools v2.27.1 and only those peaks called in both replicates were retained for downstream analysis
Assembly: mm9
Supplementary files format and content: bedfile contains Rif1 narrow and broad peaks as well as Rif1 assiciated domains called in both replicates
Supplementary files format and content: bigWigs contain either genome coverage of Rif1 ChIPseq or log2 ratios between Rif1 ChIP and input
|
| |
|
| Submission date |
Apr 03, 2023 |
| Last update date |
Jun 13, 2023 |
| Contact name |
Tobias Neumann |
| Organization name |
IMP
|
| Street address |
Campus-Vienna-Biocenter 1
|
| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE228875 |
RIF1 regulates replication origin activity and early replication timing in B cells [ChIP-seq] |
| GSE228880 |
RIF1 regulates early replication timing in murine B cells. |
|
| Relations |
| BioSample |
SAMN34058696 |
| SRA |
SRX19856737 |
