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| Status |
Public on Oct 01, 2018 |
| Title |
Ctrl-P6-2 |
| Sample type |
SRA |
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| Source name |
Ventricle
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL6/J
developmental stage: postnatal 6 days
genotype: ERRalphaHet/ERRgammaWT
Sex: not determined
datatype: sNucDrop-Seq
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
Control (ERRαHet/ERRγWT) and KO (ERRαKO/ERRγKO) mouse hearts were rapidly dissected on ice 10am – 12pm of the day. Ventricles were freshly processed or flash frozen in liquid nitrogen and subsequently kept at -80°C before nuclear isolation. Tissues were dounced 15 times with loose pestle and then 10 times with tight pestle. For nuclei purification, samples were ultracentrifuged at 25,000 rpm at 4 °C for 2 hours.
Single nucleus RNA sequencing was performed using the Drop-Seq method. Briefly, single nuclei, polyA-containing barcoded beads (ChemGenes), and droplet generation oil (Bio-Rad) were each flowed into individual input ports on a microfluidic device (uFluidix). Nuclei in droplets were lysed and hybridized to beads. Beads were isolated and subjected to reverse transcription (Thermo Fisher) and exonuclease I (NEB) treatment, followed by PCR of full length transcripts (Kapa). Final libraries were created by tagmentation of cDNA (Illumina), and amplified by PCR and purified by SPRI beads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Paired-end sequencing reads of Single nucleus RNA-seq were processed using publicly available the Drop-Seq Tools v1.12 software. Briefly, each mRNA read (read2) was tagged with the cell barcode (bases 1 to 12 of read 1) and unique molecular identifier (UMI, bases 13 to 20 of read 1), trimmed of sequencing adaptors and poly-A sequences, and aligned using STAR v 2.5.2a to the mouse (mm10, Gencode release vM13). The intronic reads were also retained for downstream analysis. A custom Perl script was implemented in the Drop-Seq Tools pipeline to retrieve both exonic and intronic reads mapped to predicted strands of annotated genes. Uniquely mapped reads were grouped by cell barcodes. Cell barcodes were corrected for possible bead synthesis errors, using the DetectBeadSynthesisErrors program from the Drop-Seq Tools v1.12 software. To generate digital expression matrix, a list of UMIs in each gene (as rows), within each nucleus (as columns), was assembled, and UMIs within ED = 1 were merged together. The total number of unique UMI sequences was counted, and this number was reported as the number of transcripts of that gene for a given nucleus.
Genome_build: mm10
Supplementary_files_format_and_content: gene expression matrix containing read counts for each gene (rows) in each cell/nucleus (columns)
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| Submission date |
Aug 14, 2018 |
| Last update date |
Oct 01, 2018 |
| Contact name |
Hao Wu |
| E-mail(s) |
haowu2@pennmedicine.upenn.edu
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| Phone |
215-573-9360
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| Organization name |
University of Pennsylvania
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| Department |
Genetics
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| Lab |
Hao Wu
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| Street address |
415 Curie Boulevard
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE118545 |
Single-nucleus transcriptomic survey of cell diversity and functional maturation in the postnatal mammalian hearts |
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| Relations |
| BioSample |
SAMN09832186 |
| SRA |
SRX4552056 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3332252_Ctrl-P6-2_gene_exonic.intronic_tagged.dge.txt.gz |
3.6 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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