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| Status |
Public on Apr 28, 2024 |
| Title |
Ehf_KO, ATACseq Rep4 |
| Sample type |
SRA |
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| Source name |
mammary gland
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| Organism |
Mus musculus |
| Characteristics |
cell type: Luminal mammary epithelial cells
tissue: mammary gland
strain: EhfKO
age: Adult, 18.5 days pregnant
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Thoracic and inguinal mammary glands were resected, lymph nodes removed, and samples collected into cold MEC media (DME-F12 supplemented with 1 mM glutamine, 5µg/ml insulin, 500ng/mL hydrocortisone, 10ng/mL Epidermal growth factor, 5% FBS). Glands were mechanically dissociated with a McIlwain tissue chopper then digested for 1 hour at 37°C in an orbital shaker with 300 U/mL collagenase, 100 U/mL hyaluronidase and 0.1 mg/mL DNAse I in MEC media containing 5% FBS. The resulting suspension was diluted in PBS with 2% FBS and cells pelleted by centrifugation. The cell pellet was further digested in 0.25% trypsin-EGTA with 0.1 mg/mL DNAse I for 3 minutes at 37°C, then diluted in PBS with 10% FBS and cells pelleted by centrifugation. The cell pellet was then digested in 5mg/mL Dispase with 0.1mg/mL DNAseI at 37°C for 5 minutes, then diluted in PBS with 2% FBS. Cells were passed through a 100 mm cell strainer to obtain a single cell suspension, then live cells counted using trypan blue. Single cells were pelleted by centrifugation, then blocked in rat γ-globulin and anti-CD16/CD32 Fcγ III/II receptor antibody with 0.1mg/mL DNAseI for 10 minutes at RT. Antibody incubations were performed at 4°C for 25 minutes. Antibodies used for Fluorescence-activated cell sorting (FACS) were: APC anti-mouse CD31, APC anti-mouse CD45 Antibody, APC anti-mouse TER-119/Erythroid Cells, Pacific Blue anti-mouse CD24 and FITC-anti-mouse CD29. Cells were diluted in PBS with 2% FBS then pelleted by centrifugation, and 0.2mg/ml 7-Aminoactinomycin D added to exclude dead cells. The cell suspension was then filtered through a 40 µm cell strainer and luminal mammary epithelial cells (Ter119–CD31−CD45–CD24+CD29Lo) sorted on a FACS Aria using a 100 µm nozzle.
Nuclei were extracted from 50,000 cells per sample by resuspending in 50 µl of ATAC-seq lysis buffer (10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% IGEPAL, 0.01% Digitonin) and incubating on ice for 3 minutes followed by washing with 10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20. Samples were then centrifuged at 500 g for 10 minutes at 4°C and the supernatant discarded. Transposition of the DNA and ligation of adapters was performed using the Illumina Tagment DNA TDE1 Enzyme and buffer kit (20034197) as per the manufacturer’s instructions. Briefly, DNA was digested by incubation with Tn5 Transposase enzyme at 37°C for 30 minutes. Transposed DNA was then purified using the Qiagen MinElute reaction cleanup kit (Qiagen, 28204), and partially PCR-amplified using Nextera custom barcoded primers for 5 cycles. To reduce GC-bias, the number of additional PCR cycles required was determined to be 9 cycles (14 cycles in total) by using a SYBR Green q-RT-PCR reaction and calculating the number of cycles corresponding to 1/3 of the maximum fluorescent intensity. Libraries were then purified by AMPure XP magnetic bead purification (Beckman Coulter A63880). DNA quantity and quality was assessed using the Tapestation 2000 system (Agilent).
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
10x Genomics
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| Data processing |
ATAC-seq Reads were aligned to the GRCm39/mm39 build of the mouse reference genome by using the Subread aligner in the Subread package (version 2.0.6). Only uniquely mapped reads were included. Peaks in each individual sample were called by using Homer (v4.11) with a false discovery rate (FDR) cutoff of <1e-5. The peaks from all the samples were then merged by taking unions of the overlapping peaks; This produced 195818 merged regions. The mapped reads in the individual samples were then assigned to the merged regions by using featureCounts. Multi- assignment of a single read was allowed. Merged regions were required to have at least 10 CPMs in at least 4 samples to be included in the analysis. Read counts were converted to log2-CPM, quantile normalised and precision weighted with the voom function of the limma package. Batch effect was removed by using the removeBatchEffect function in limma. Sample clustering was performed based on multi-dimensional scaling.
A correlation analysis was performed between the 352 DE genes identified in the Avd-G population from the scRNA-seq analysis and the regions of differentially accessible chromatin identified in the luminal cell population from EhfWT and EhfKO samples. Out of the 352 DE genes, 331 genes commonly found between the scRNA-seq data and the ATAC-seq data were included in the correlation analysis. Pearson correlation was performed by first calculating the log2 CPM values of promoter regions of all genes in the ATAC-seq data. The promoter regions were defined as the 5kb up and 2kb down region around the TSS of each gene in the mm39 RefSeq gene annotation. A scatter plot was generated to show the log2 fold changes in scRNA-seq and ATAC-seq data. Gene read coverage track figures show the coverage levels (CPM) of the ATAC-seq reads in the promoter regions of the selected genes. The selected genes have fold changes ≥1.2 in scRNA-seq Avd-G data and in ATAC-seq luminal data. The denominators for calculating CPM values are the number of mapped reads in each library. 50-bp bins in the promoter regions were used.
Assembly: mm39
Supplementary files format and content: Tab-delimited text files include log2-CPM values for each library after the filtering step. The supplementary file, raw_read_counts.txt, includes raw read counts for merged peak regions in all the libraries before filtering.
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| Submission date |
Mar 05, 2024 |
| Last update date |
Apr 28, 2024 |
| Contact name |
Wei Shi |
| E-mail(s) |
[email protected]
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| Organization name |
Olivia Newton John Cancer Research Institute
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| Department |
Bioinformatics and Cancer Genomics
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| Street address |
Level 5, ONJ Cancer Centre, 145 Studley Rd
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| City |
Heidelberg |
| State/province |
VIC |
| ZIP/Postal code |
3084 |
| Country |
Australia |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE260878 |
EHF is an essential regulator of mammary alveolar lineage differentiation and putative suppressor of breast tumorigenesis |
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| Relations |
| BioSample |
SAMN40261863 |
| SRA |
SRX23842679 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8127358_KO4_Luminal_S16_log2CPM.tsv.gz |
432.3 Kb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
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