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| Status |
Public on Apr 08, 2021 |
| Title |
Sham scRNA rep 1 |
| Sample type |
SRA |
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| Source name |
Heart tissue (non-cardiomyocyte cells)
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| Organism |
Mus musculus |
| Characteristics |
heart surgery: Sham
treatment: Vehicle
type of experiment: Single cell RNA seq
cell type: Heart tissue (non-cardiomyocyte cells)
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| Treatment protocol |
n/a
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| Growth protocol |
n/a
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Briefly, after proper anesthesia level was reached, thoracotomy was performed, and mouse heart was isolated. The isolated heart was cannulated and perfused with perfusion buffer (120.4 mM NaCl, 14.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 10 mM Na-HEPES, 4.6 mM NaHCO3, 30 mM taurine, 10 mM 2,3-butanedione monoxime, and 5.5 mM glucose, pH 7.0) in a Langendorff perfusion system (Radnoti 120108EZ) for 5-10 minutes at 37 °C. The cannulated heart was then digested by digestion buffer (perfusion buffer with 300 units/mL collagenase II (Worthington Biochemical) and 50 μM CaCl2) for about 10 min at 37 °C. At the end of digestion, the atria and great vessels were removed and the ventricular tissue was transferred to and gently teased into small pieces in stop buffer (perfusion buffer with 10% fetal bovine serum) at 37 °C. After gently pipetting, cell suspension was passed through a 250uM strainer in a falcon tube and then at 30xg for 3 minutes at room temperature (RT). Then, the supernatant - containing most of the non-cardiomyocytes (CMs) - was divided from the pellet (containing the CM fraction). The non-CM fraction was centrifuged again at 30xg for 3 minutes at RT and the supernatant kept. The supernatant was then filtered with a cell strainer (70um) and finally centrifuged at 400xg for 3 minutes at RT for eliminating debris. The non-CM pellet was finally resuspended in 1mL cold PBS 0.5% BSA. 30k cells were counted with trypan blue using a hemocytometer and then used for subsequent 10X Genomics Chromium single cell RNAseq preparation. For single cell ATAC, 500k isolated and purified non-CMs were resuspended in 100uL lysis buffer (Tris-HCl 10mM pH 7.4, NaCl 10mM, MgCl2 3mM, Tween-20 0.1%, P40 0.1%, Digitonin 0.01%, BSA 1% in Nuclease-free water), pipetted 10 times and kept on ice for 5 minutes. Nuclei were then washed 1mL 1X PBS with 1% BSA and then centrifuged at for 5 minutes at 4C. The nuclei pellet was resuspended in 1mL 1X PBS with 1% BSA and filtered with a 10uM strainer (pluriSelect #43-50010-00). Nuclei were then counted with DAPI using a hemocytometer and finally 30k non-CM nuclei used for subsequent 10X Genomics Chromium single cell ATACseq preparation. The CM-fraction was, after the first centrifugation, centrifugated again at 30xg for 3 minutes at RT in stopping buffer and the supernatant was discarded. The CM pellet was finally centrifuged at 400xg for 3 minutes in stopping buffer at RT and after discarding the supernatant, the cell pellet was lysed in Qiazol (miRNeasy kit - Qiagen) for subsequent RNA extraction.
Single-cell droplet libraries from the non-CM cell suspension (2x Sham, 2x TAC-Veh, 2x TAC-JQ1 and 2x TAC-JQ1 withdrawn) were generated in the 10X Genomics Chromium controller according to the manufacturer’s instructions in the Chromium Single Cell 3′ Reagent Kit v.2 User Guide. Additional components used for library preparation include the Chromium Single Cell 3′ Library and Gel Bead Kit v.2 (PN-120237) and the Chromium Single Cell 3′ Chip kit v.2 (PN-120236). Libraries were prepared according to the manufacturer’s instructions using the Chromium Single Cell 3′ Library and Gel Bead Kit v.2 (PN-120237) and Chromium i7 Multiplex Kit (PN-120262). Final libraries were sequenced on the NextSeq 500 (Illumina) for a quality control run and then on the NovaSeq (Illumina) for deeper sequencing. All the 8 samples were pooled and sequenced in one single lane. Sequencing parameters were selected according to the Chromium Single Cell v.2 specifications. All libraries were sequenced to a mean read depth of at least 50,000 total aligned reads per cell. Raw sequencing reads were processed using the Cell Ranger v.2.2.0 pipeline from 10X Genomics. In brief, reads were demultiplexed, aligned to the mouse mm10 genome and UMI counts were quantified per gene per cell to generate a gene-barcode matrix. Data from the 8 samples were aggregated and normalized to the same sequencing depth, resulting in a combined gene-barcode matrix of all samples.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
cellranger (version 3.0.2) mkfastq with default parameters to demultiplex raw base calls
cellranger (version 3.0.2) count with default parameters for alignment, filtering, barcode counting, UMI counting, and cell calling. See https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/algorithms/overview for details
Genome_build: Mm10
Supplementary_files_format_and_content: We provide barcodes.tsv files, matrix.mtx files and features.tsv files (for single cell RNA seq) or peaks.bed files (for single cell ATAC seq) generated by the 10x Genomics cellranger command. We provide BigWig files for PROseq and 4C. We provide bed files and bigWig files for ChIPseq. We provide excel files for differential expression analysis with Reads Per Kilobase of exon per Megabase of library size (RPKM) for every gene.
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| Submission date |
Aug 07, 2020 |
| Last update date |
Apr 08, 2021 |
| Contact name |
Pawel Przytycki |
| E-mail(s) |
pawel.przytycki@gladstone.ucsf.edu
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| Organization name |
Gladstone Institutes
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| Department |
GIDB
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| Lab |
Katie Pollard
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| Street address |
1650 Owens Street
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| City |
San Francisco |
| State/province |
California |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE155882 |
A Transcriptional Switch Governs Fibroblast Activation in Heart Disease |
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| Relations |
| BioSample |
SAMN15760101 |
| SRA |
SRX8909980 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4715045_Sham_scRNA_rep1_barcodes.tsv.gz |
2.4 Mb |
(ftp)(http) |
TSV |
| GSM4715045_Sham_scRNA_rep1_features.tsv.gz |
272.8 Kb |
(ftp)(http) |
TSV |
| GSM4715045_Sham_scRNA_rep1_matrix.mtx.gz |
59.2 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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