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| Status |
Public on Jan 14, 2024 |
| Title |
CHIC-Seq, Pbrm1-WT1 |
| Sample type |
SRA |
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| Source name |
D4 WT primary uterine stromal cell Pbrm1
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| Organism |
Mus musculus |
| Characteristics |
cell type: primary uterine stromal cell
genotype: WT
time: Day 4 of pregrancy
antibody: Pbrm1
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| Treatment protocol |
Mate with stud male for pregrancy.
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| Growth protocol |
The mice were housed in NIDDK mouse core facility, in accordance with guidelines of the Animal Care and Use Committee of the National Institutes of Health under a Division of Intramural Research, NIDDK approved animal study protocols. Uteru isolated from 8-12 weeks aged .
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5-10 pseudopregnant D4 WT mouse uterine horns were cut into small pieces (2–3 mm). Tissue pieces were first digested in 5 ml fresh medium (HBSS antibiotic; Gibco) containing 6 mg/ml dispase (Gibco) and 25 mg/ml pancreatin (Sigma), and then incubated in fresh medium (3 ml) containing 0.5 mg/ml collagenase (Sigma) at 37°C for 30 min. The digested cells were passed through a 70-lm filter to obtain the stromal cells. Cells were plated at 60-mm dishes, containing DMEM and Ham F-12 nutrient mixture (1:1) (Gibco) with 10% charcoal-stripped fetal bovine serum (FBS) and antibiotic. After 4 h, the medium was replaced with fresh medium (DMEM/F-12, 1:1) with 10% FBS. 2 hours later, wash twice with PBS gently for the preparation of CHIC-Seq samples.
Small cell number ChIC-seq assays were performed as previous reported. ChIC-seq libraries were constructed using 50,000 mouse primary uterine stromal cells. Cells were fixed with 1% formaldehyde solution at RT for 5 minutes. Reaction was terminated by adding 1:10 volume of 1.25 M glycine. The fixed cells were kept in -80 ℃ after three washes with ice cold PBS. For ChIC reaction and library preparation, cells were incubated and rinsed with 500 ml antibody binding buffer (1xTE pH 7.5+150 mM NaCl+0.1% Trition X100) twice and re-suspended in 100 ml antibody binding buffer. To prepare ProteinA-MNase and antibody complex, Pbrm1 (Abcam, ab196022), Brg1 (Abcam, ab110641), bounded PA-MNase (Ab+PA-MNase) and were incubated at a molecular ratio of 1:2 on ice for 30 min in 50 ml antibody binding buffer. This complex mixed with 50 ml resuspended cells and incubated for 1.5 h on ice. Cells were washed three times with 1 ml wash buffer (1xTE pH 7.5+400 mM NaCl+1% Triton X-100) and rinsed with 400 ml rinsing buffer (10 mM Tris pH 7.5+10 mM NaCl+0.1% Triton X-100). The MNase digestion was initiated by re-suspending rinsed cells in 40 ml reaction buffer (20 mM Tris, pH 7.4, 10 mM NaCl, 2 mM CaCl2, 0.1% Tx100) and incubated at 37 ℃ for 3 min. The reaction was stopped by adding 80 ml stop buffer (20 mM Tris, pH 8.0, 10 mM EGTA, 20 mM NaCl, 0.2% SDS) and 1 ml proteinase K, then incubated at 65 ℃ for overnight. DNAs were purified by phenol-chloroform extraction. The purified DNA was used for library preparation. For all ChIC-seq libraries, PCR was performed for 13-15 cycles and DNA fragments from 200 bp to 500 bp were isolated and sequenced on NovaSeq 6000 (Illumina).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Baf_peaks.txt
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| Data processing |
removing adapter sequences with cutadapt
aligning the reads with Bowtie2 against Mus musculus genome sequence
filter for uniquely mapped and non-duplicated reads
call peaks with macs2
Assembly: GRCm38.p5
Supplementary files format and content: peaks called in txt files
Library strategy: CHIC-seq
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| Submission date |
Apr 11, 2023 |
| Last update date |
Jan 14, 2024 |
| Contact name |
qiliang Xin |
| Organization name |
NIH
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| Department |
NIDDK
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| Lab |
LCDB
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| Street address |
50 South Dr
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20894 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE229368 |
Stromal Pbrm1 mediates chromatin remodeling necessary for embryo implantation in mouse uterus [CHIC-Seq] |
| GSE229370 |
Stromal Pbrm1 mediates chromatin remodeling necessary for embryo implantation in mouse uterus |
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| Relations |
| SRA |
SRX19926138 |
| BioSample |
SAMN34138328 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7159354_Baf180_1_peaks.txt.gz |
658.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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