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GEO help: Mouse over screen elements for information. |
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Status |
Public on Aug 28, 2016 |
Title |
1772099-228_F03 |
Sample type |
SRA |
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Source name |
thoracic ganglion
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Organism |
Mus musculus |
Characteristics |
cell type: sympathetic neuronal cells Sex: F age: p33 celltype identifier: Unclassified
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Growth protocol |
R26R-Tomato (Jackson Laboratory) mice were crossed to the Wnt1-Cre (PS Danelian et at., Current biology (1998) 8:1323-1326) to allow delineation of sympathetic ganglia against surronding tissue. Animals were kept in cages in groups, with food and water ad libitum, under 12-h light-dark cycle conditions.
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Extracted molecule |
polyA RNA |
Extraction protocol |
Sympathetic ganglia (SG) were collected in freshly oxygenated artificial cerebral spinal fluid (ASCF) kept on ice. SG were transferred into a 3cm plastic dish containing 2.7mL of 37°C digestion solution (500μL TrypLE™ Express (Life Technologies), 2000μL Papain solution (Worthington Biochemical, 25U/ml), 100 μL DNAseI (Worthington Biochemical, 1 mM) and 100 μL Collagenase/Dispase (Roche) 20 mg/ml). SGs were mechanically triturated every 30 min using glass pasteur pipettes. After 1h incubation at 37°C, 100 μL fresh Collagenase/Dispase (20mg/mL) solution was added and SGs were ripped open by using fine forceps. The solution was filtered using a 40μm cell strainer (FALCON) and collected in a 15 ml plastic tube. The digestion solution was diluted in 3ml ACSF and centrifuged at 100xg for 4 min at 4°C. The supernatant was removed and the pellet resuspended in 0.5 mL ASCF and 0.5 mL Neurobasal-A medium (Gibco). The cell suspension was transferred onto 80μl of Optiprep Density Solution (Sigma), 460 μL of ACSF and 460 μL Neurobasal media with supplements, and centrifuged at 100xg for 10min at 4°C and the solution concentrated to 100 μL by removing the supernatant. 10μL DNaseI was added to avoid cell clustering. Cell density wasa djusted to 400-500 cells/μL. The Fluidigm C1 Autoprep System microfluidic chip (medium size) was used to capture cells (Zeisel et al., Science (2015) 6;347(6226):1138-42). Cells at the capture sites were visually inspected and only single healthy cells were selected for library preparation. Cell barcoding and fragmentation were performed in a single step using Tn5 DNA transposase as described previously (ibid.) but using a different Binding and Blocking buffer (10mM Tris, 250mM NalCl, 5mM EDTA, 0.5% SDS).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Description |
SingleCell144
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Data processing |
Read processing was performed as described (Islam et al., Nat Methods (2014) 11(2):163-6), except that we removed any RNA molecule (i.e. Unique Molecular Identifier) supported only by a single read ("singleton molecules"). This removed a large number of false positive molecules, artefacts that can arise by sequencing error, PCR-induced mutations or translocations and cross-contamination. The first 6 bases of each read represent the random Unique Molecular Identifier used for molecule counting. After follows three or more Gs, stemming from the template switching at the mRNA 5' end during first strand cDNA sythesis. We rejected cells that had less than 2000 mRNA molecules, more than 26,000 mRNA molecules (putative doublets) as well as cells that formed a separate cluster with no specific gene expression (putative damaged or dead cells). Genes that were detected at less than 4 molecules in the whole datasets were eliminated. Genome_build: UCSC mm10 Supplementary_files_format_and_content: Tab-delimited table of total number of detected mRNA molecules from each gene in each cell identified by the "Title" field in Samples list.
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Submission date |
Mar 02, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Sten Linnarsson |
Organization name |
Karolinska Institutet
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Department |
Medical Biochemistry and Biophysics
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Lab |
Molecular Neurobiology
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Street address |
Scheeles väg 1
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City |
Stockholm |
ZIP/Postal code |
171 65 |
Country |
Sweden |
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Platform ID |
GPL13112 |
Series (1) |
GSE78845 |
Single cell transcriptome analysis of mouse thoracic sympathetic ganglia |
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Relations |
BioSample |
SAMN04529273 |
SRA |
SRX1610727 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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