|
| Status |
Public on Jun 01, 2023 |
| Title |
peritoneal macrophages - IRF8_rep2_batch1 |
| Sample type |
SRA |
| |
|
| Source name |
FACS-sorted microglia
|
| Organism |
Mus musculus |
| Characteristics |
gender: Female
strain: IRF8-EGFP
chip antibody: GFP (Abcam, ab290)
|
| Treatment protocol |
Brain cells were crosslinked with 1% PFA/PBS for 10 min prior to sorting in case of transcription factor ChIP
|
| Growth protocol |
In vivo microglia were non-enzymatically isolated from a 3-month-old WT and IRF8KO brain and enriched by cell sorting.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cells/nuclei were immobilized with Con-A beads and incubated with an antibody overnight. The immunocomplexes were further incubated with protein A-fused micrococcal nuclease (pA-MNase), then digested for 30 min in the presence of 3mM Ca2++ (Ref: PMID: 28079019).
If needed, the ChIPped DNA was de-crosslinked and purified by phase extraction. The DNA was filtered with 0.5x AMPure beads to remove unwanted large fragments. Purified ChIPed DNA was then processed to construct a library using SMARTer® ThruPLEX DNA-Seq Kit (TAKARA Bio USA, CA) as the manufacturer’s protocol except PCR cycles to avoid amplifying long fragments (13 cycles, 10s for extension).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
2x35 paired-end sequence reads
|
| Data processing |
Basecalls performed using bcl2fastq without the acceptance of index mismatch
Sequenced reads were trimmed for the adaptor and low-quality sequences with TrimGalore, then mapped to mm10 whole genome using bowtie2 Bowtie2 with --local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700 --no-overlap --no-dovetail options
Bigwig files were generated using deeptools bamCoverage with -bs 10 --normalizeUsing RPGC --extendReads options after merging all bam files by each genotype.
Peaks were called using MACS2 with --keep-dup all -q 0.01 –max-gap 640 options
Assembly: mm10
Supplementary files format and content: Bigwig (.bw) files consist of RPGC-normalized average ChIP signal by genotype.
Supplementary files format and content: Peak files (.xls) contain peak regions and tag density generated by MACS2
|
| |
|
| Submission date |
May 01, 2023 |
| Last update date |
Jun 01, 2023 |
| Contact name |
Keita Saeki |
| E-mail(s) |
keita.saeki@nih.gov
|
| Organization name |
NIH/NICHD
|
| Street address |
6 Center Drive
|
| City |
Bethesda |
| State/province |
Maryland |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE231402 |
Genome-wide profiles of IRF8 and PU.1 bindings, and H3K4me1 and H3K27ac histone marks in wild-type and IRF8-knockout microglia [CHIP-seq] |
| GSE231406 |
IRF8 defines the epigenetic landscape in postnatal microglia, thereby directing their transcriptome programs |
|
| Relations |
| BioSample |
SAMN34507561 |
| SRA |
SRX20177991 |
