|
| Status |
Public on Sep 13, 2024 |
| Title |
Young,Uterus,rep2 [Y30_input] |
| Sample type |
SRA |
| |
|
| Source name |
Uterus
|
| Organism |
Mus musculus |
| Characteristics |
tissue: Uterus
cell line: C57BL/6Babr
cell type: Uterine stromal Cells
genotype: Wild-type
treatment: Young
|
| Treatment protocol |
Cells were isolated from uteri of young and aged females.
|
| Growth protocol |
Uterine stromal cells were isolated as described previously (Woods et al., 2017). In brief, uteri from E3.5 mice were slit longitudinally and disaggregated with 2.5% pancreatin and 0.5% trypsin (type III) in Hank’s basic salt solution-calcium/magnesium free for 1.5 hour on ice. The digested tissue was vortexed and the medium was removed and the remaining tissue washed twice in HBSS (discarding luminal epithelial cells) and incubated 30 min at 37oC in HBSS containing 0.007% collagenase, 0.02% DNase, 0.008% protease with vortexing every 5 min. Dissociated tissues were then triturated, filtered through a 70μm cell strainer (BD Biosciences) and spun at 3000 rpm for 5min at 4ºC. Cell pellets were resuspended in standard growth medium and plated.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Histone ChIP was performed on pellets of 200,00 uterine stromal cells. Chromatin was digested with micrococcal nuclease and ChIP performed with antibodies against H3K4me3 (Abcam ab8580) and H3K9me3 (Abcam ab8898). 10% of chromatin was kept back and sequenced as Input. Following the chromatin immunoprecipitation procedure, DNA was isolated using proteinase K digestion followed by clean-up using the QiaQuick PCR purification kit (Qiagen 28104).
Input and ChIP DNA was used for library build with the NEBNext Ultra II DNA library prep kit (Illumina E7645)
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
ChIP-seq of uterine stromal cells from young female, replicate 4
Y30_input
|
| Data processing |
Indexed libraries were pooled and sequenced on an Illumina HiSeq2500 sequencer, using a 50bp paired-end protocol. Read depth per sample was between 7.7M and 20.4M reads.
Raw fastq data were mapped to the Mus musculus GRCm38 genome assembly with Bowtie2
Peaks were called using the MACS peak caller in SeqMonk. Differential enrichment was determined using the DESeq2 package or LIMMA statistics in SeqMonk.
Bigwig files were generated from the bam files using bamCoverage from deeptools with options --normalizeUsing CPM --minMappingQuality 20
Bedgraph files were exported from Seqmonk and Bigwig files were generated from the bam files using bamCoverage from deeptools with options --normalizeUsing CPM --minMappingQuality 20
Assembly: GRCm38
Supplementary files format and content: Bigwig and Bedgraph files are provided for each chip relative to input
|
| |
|
| Submission date |
May 14, 2024 |
| Last update date |
Sep 13, 2024 |
| Contact name |
Myriam Hemberger |
| E-mail(s) |
myriam.hemberger@ucalgary.ca
|
| Organization name |
University of Calgary
|
| Street address |
3330 Hospital Dr NW
|
| City |
Calgary |
| State/province |
Alberta |
| ZIP/Postal code |
T2N 4N1 |
| Country |
Canada |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE267479 |
H3K4me3 and H3K9me3 histone modification profiles of uterine stromal cells from young and aged mouse females |
|
| Relations |
| BioSample |
SAMN41396976 |
| SRA |
SRX24550153 |
