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| Status |
Public on May 02, 2018 |
| Title |
Sox2-GFP+ve alone 2 |
| Sample type |
SRA |
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| Source name |
subventricular zone
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| Organism |
Mus musculus |
| Characteristics |
transgenic line: aSMACreERt2::Rosa26-tdTomato x B6;129S-Sox2tm2Hoch/J
facs strategy: tdTomato-negative/GFP-positive
# facs sorted cells: 6000
library preparation: 10X Genomics Single Cell RNAseq v2
strain background: C57/Bl6(3/4) x 129S1SvlmJ(1/4)
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
The lateral wall of lateral ventricles (SVZ) from adult mice (n = 5) were microdissected and enzymatically dissociated with Papain (Worthington, 30 U/mL with 330µg/mL L-Cysteine in HBSS) at 37 °C for 30 mins with trituration every 10 minutes. Cell suspensions were then filtered through a 40µm Nylon cell strainer to obtain a single cell suspension. Liberated cells were re-suspended in 0.5% BSA in HBSS before FACS purification. For FACS purification/sorting, a stringent initial gate was used to exclude debris, and cell doublets. Wildtype cells were used as negative control to determine gates for detection of GFP and tdTOMATO-fluorescence, then cells from αSMACreERT2::tdTomato/Sox2::GFP mice were purified according to these gates. Cortical tissue (devoid of ependymal cells) was dissociated and sorted as above in order to determine gates distinguishing two subpopulations of αSMACreERT2::tdTomato+ve cells from SVZ tissue. All cells were processed according to 10x Genomics ChromiumTM Single Cell 3’ Reagent Guidelines (v2 Chemistry; https://support.10xgenomics.com/single-cell-gene-expression). Briefly, cells were partitioned into nanoliter-scale Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology.
Primers containing (i) an Illumina R1 sequence, (ii) a 16 bp 10x barcode, (iii) a 10 bp Unique Molecular Identifier (UMI) and (iv) a poly-dT primer sequence were incubated with partitioned cells resulting in barcoded, full-length cDNA from poly-adenylated mRNA. Silane magnetic beads were used to remove leftover biochemical reagents/primers, then cNDA was amplified by PCR. Enzymatic Fragmentation and Size Selection was used to optimize cDNA amplicon size prior to library construction. R1 (read 1 primer sequence) were added during GEM incubation, whereas P5, P7, a sample index (i7), and R2 (read 2 primer sequence) were added during library construction via End Repair, A-tailing, Adaptor Ligation and PCR. Quality control and quantification was performed using a Kapa Library Quantification qPCR kit.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Reads were processed using the 10X Genomics Cell Ranger Single Cell 2.0.0 pipeline with default and recommended parameters, as previously described (Zheng et al., 2017). FASTQs generated from Illumina sequencing output were aligned to the mouse GRCm38.p5 (primary assembly) reference genome using the STAR algorithm. Next, Gene-Barcode matrices were generated for each individual sample by counting unique molecular identifiers (UMIs) and filtering non-cell associated barcodes. Finally, all three samples were aggregated, with intermediary depth normalization, to generate a gene-barcode matrix containing 1700 barcoded cells and gene expression counts. This output was then imported into the Seurat (v1.4.0.15) R toolkit for quality control and downstream analysis of our single cell RNAseq experiment. All functions were run with default parameters, unless specified otherwise. Low quality cells (<400 genes/cell and <3 cells/gene) were discluded from the overall experiment. Gene expression was log normalized to a scale factor of 10 000.
Genome_build: mm10 (GRCm38.p5)
Supplementary_files_format_and_content: Tab-delimited table of Seurat processed and scaled single cell barcoded mRNA counts
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| Submission date |
Jun 21, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Jeff Biernaskie |
| E-mail(s) |
jeff.biernaskie@ucalgary.ca
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| Organization name |
University of Calgary - Faculty of Veterinary Medicine
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| Street address |
3300 Hospital Dr NW
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| City |
Calgary |
| State/province |
Alberta |
| ZIP/Postal code |
T2N 4N1 |
| Country |
Canada |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE100320 |
Single-Cell Transcriptomics and Fate Mapping of Ependymal Cells Reveals an Absence of Neural Stem Cell Function |
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| Relations |
| BioSample |
SAMN07261720 |
| SRA |
SRX2941876 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2677819_sample_3_barcodes.tsv.gz |
5.4 Kb |
(ftp)(http) |
TSV |
| GSM2677819_sample_3_genes.tsv.gz |
212.7 Kb |
(ftp)(http) |
TSV |
| GSM2677819_sample_3_matrix.mtx.gz |
4.7 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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