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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Nov 11, 2018 |
| Title |
P0 whole heart, biological replicate 1, technical replicate 3 |
| Sample type |
SRA |
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| Source name |
P0 whole heart
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| Organism |
Mus musculus |
| Characteristics |
internal sample id: JD131
tissue: whole heart
developemental stage: P0
background strain: C57BL6+ICR
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| Treatment protocol |
Primary cardiac cells were prepared by dissecting and mincing neonatal mouse hearts. The tissue was then incubated in trypsin for a total of 30 minutes in a shaking water bath at 37˚C. Supernatant was strained and transferred to a flask containing cold growth media every 5 minutes--6 fractions total. Cells were spun at 500x G for 5 minutes at 4˚C and resuspended in cold growth media.
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| Growth protocol |
Primary cardiac cells were harvested from neonatal mouse hearts. Mice were housed in a standard facility 12hr light/dark cycle, 70-75˚F, and ~50% humidity.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single cell suspension was washed with 1X PBS (0.01% BSA) before Drop-Seq procedure. Drop-seq was performed as described previously (Macosko et al., 2015) with small modifications. Briefly, droplets were generated using microfluidic devices, which encapsulated single cells and barcoded beads (ChemGenes Corporation, Wilmington, MA, catalog number Macosko201110). Beads were collected after the breakage of individual droplets using perfluorooctanol (Sigma-Aldrich, St. Louis, MO, catalog number 370533). RNA hybridized on the barcoded beads was reverse transcribed using Maxima H minus reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, catalog number EP0751) and cDNA was amplified with 13 cycles (KAPA HotStart ReadyMix, Kapa Biosystems, Wilmington, MA, catalog number KK2602).
In-house Tn5 transposase was used to insert sequencing adapters and fragmentate cDNA. Illumina Nextera adapters i5 and i7 were used to amplify the fragmented cDNA with 12 cycles (KAPA HiFi PCR Kits, catalog number KK2102). Agarose gel size selection was performed to recover fragments with length of 400 to 600 bp.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
JD131-B
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| Data processing |
BCL files generated by Illumina sequencing systems were demultiplexed and converted to standard FASTQ files using bcl2fastq (version 2.17.1.14).
After demultiplexing, reads (read 1) with more than one low quality bases (Phred quality score less 10) in barcode or UMI regions were discarded. PolyA tails (read 2) longer than 6 bases were trimmed before mapping.
For Drop-seq, read 1 contains barcode (basepairs 1-12) and UMI (basepairs 13-20).
Reads were aligned to mouse mm10 genome assembly using STAR (version 2.5.3a) with default parameters. Secondary and repetitive alignments were removed. Only unique alignments were kept for downstream analyses.
Cell barcodes within one edit distance were collapsed together. UMI-based PCR duplicate removal was performed using the directional method of UMI-tools with default parameters (version 0.5.3). An inflection point of cumulative number of reads of cells in each library was calculated to determine the number of cells in each experiment. Gene expression matrices were generated using featureCounts (version 1.5.3). The gene annotation used is based on Ensembl release 84 (GENCODE Gene Set M9).
Genome_build: mm10
Supplementary_files_format_and_content: Raw UMI count matrices representing gene expression values (comma-separated) were generated as described above. Each column represents a single cell, each row represents a single gene.
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| Submission date |
Jul 27, 2018 |
| Last update date |
Nov 12, 2018 |
| Contact name |
Gary Hon |
| Organization name |
UT Southwestern
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| Department |
Department of Obstetrics and Gynecology
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| Lab |
Hon Lab
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| Street address |
5323 Harry Hines Blvd.
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| City |
Dallas |
| ZIP/Postal code |
75390 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE117784 |
Reprogram-Seq: A platform for single-cell combinatorial reprogramming [I] |
| GSE117795 |
Reprogram-Seq: A platform for single-cell combinatorial reprogramming |
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| Relations |
| BioSample |
SAMN09727412 |
| SRA |
SRX4475963 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3308723_expr_readcount_JD131-B.csv.gz |
831.1 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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