|
| Status |
Public on Oct 18, 2023 |
| Title |
WT B cells H3K27me3 ChIP DMSO Rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Splenic B cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: MACS-purified splenic B cells
strain: C57BL/6
genotype: WT
chip antibody: H3K27me3 (07-449, lot 3574311)
treatment: DMSO
|
| Treatment protocol |
DMSO or 1 uM GSK343 for 48 hours
|
| Growth protocol |
Splenic B cells were purified from 6-8 week old mice using MACS and CD43 microbeads (Miltenyi biotec). The cells were treated with either DMSO or 1 uM GSK343 (5x10^6 cells/mL) for 48 hours in RPMI (Wisent) supplemented with 10% (v/v) FBS, 100 IU penicillin, 100 ug/mL streptomycin, 2 mM glutamine, 55 uM 2-BME, 2ng/mL BAFF and 2ng/mL IL-4 (BioLegend).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Harvested cells were washed with PBS and fixed with 1% formaldehyde/PBS for 5 min at RT. The reaction was quenched with 0.125 M glycine for 5 min at RT. Fixed cells were washed with PBS and lysed to isolate chromatin. Chromatin was sonicated with Bioruptor Pico (Diagenode) to < 500 bp. Sonicated chromatin was diluted ten-fold and pre-cleared with Dynabeads Protein G (ThermoFisher). A small volume (5%) was saved as input. The remainder was incubated with 4 ug anti-H3K27me3 antibody per 30 ug chromatin over night. Dynabeads Protein G was added and incubated for 2 hours. Immunoprecipitated chromatin-protein complexes were washed and decrosslinked over night at 65 C. Input and ChIP DNA were treated with RNaseA and proteinase K, column purified (NEB T1030S), and quantified with Qubit (ThermoFisher).
Libraries were prepared with NEBNext Ultra II DNA kit (E7645S) and NEBNext Multiplex Oligos (E7600S), following the manufacturer's recommendations. Size selection and purification were performed with Ampure XP beads (Beckman Coulter).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Base calls and demultiplexing were performed using bcl2fastq
Paired reads were aligned to mm10 using bowtie2/2.4.2 using --sensitive-local
Concordantly mapped reads were obtained from all mapped reads using samtools/1.12 -f 0x2
Broad peaks were called using macs2/2.2.7.1 using -f BAMPE --broad and pooling biological duplicates together
Called broad peaks were then filtered to remove mm10 blacklist regions (ENCODE) using bedtools/2.29.2 intersect -v
bedtools/2.29.2 intersect -v OR -u was used to identify unique or common peaks in pairwise comparisons, respectively
Coverage tracks (excluding any blacklist regions) were generated for each biological sample using bamCoverage (deeptools/3.5.1) --normalizeUsing RPGC -e -bl $/path/to/blacklist/file. Csaw/1.34 R package was used to calculate normalization values for ChIP libraries, which were used as input values for --scaleFactor option.
Data was visualized using deeptools/3.5.1 and IGV/2
Assembly: mm10
Supplementary files format and content: bw: RPGC coverage file from bamCoverage; broadPeak: peak calls from macs2
|
| |
|
| Submission date |
Jul 19, 2023 |
| Last update date |
Oct 18, 2023 |
| Contact name |
Frederick Andrew Dick |
| E-mail(s) |
fdick@uwo.ca
|
| Organization name |
Western University
|
| Department |
Pathology
|
| Lab |
A4-VRL LRCP
|
| Street address |
790 Commissioners Road E
|
| City |
London |
| State/province |
ON |
| ZIP/Postal code |
N6A 4L6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE198158 |
EZH2 inhibition with GSK343 reduces H3K27me3 deposition at various repetitive elements in splenic B cells [ChIP-Seq] |
| GSE198232 |
Cytosolic pattern recognition receptors respond to EZH2-inhibition induced viral mimicry response that eliminates splenic B cells |
|
| Relations |
| BioSample |
SAMN36632071 |
| SRA |
SRX21142444 |
