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Status |
Public on Dec 14, 2018 |
Title |
C1-1772117-115_E03 |
Sample type |
SRA |
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Source name |
LT-NES SAI2 cell
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Organism |
Homo sapiens |
Characteristics |
cell type: long-term self-renewing neuroepithelial-like stem cells
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Growth protocol |
Cells were grown in maintanence medium based on DMEM F12 Glutamax Medium (GIBCO, LifeTechnologies) supplemented with N2 (1:100, GIBCO, LifeTechnologies), B27 (1:1000, GIBCO, LifeTechnologies), and a combination of growth factors hEGF (10 ng/ml, R&D) and FGF2 (10 ng/ml, R&D).
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Extracted molecule |
polyA RNA |
Extraction protocol |
Cells were trypsinized with TrypLE Select and cell resuspensions were kept in maintenance medium with albumin from bovine serum until loading on the Fluidigm chip. Fluidigm C1 Autoprep System Cells medium (10-17 micrometer) microfluidic was used to capture the cells. 14 microlitre of cell suspension (approx. 800 cells/microlitre) was mixed with 7 microlitre C1 Suspension Reagent after filtering. Single-cells were then captured for 30 min at 4°C using the “Cell Load (1772x/1773x)” script. The protocol for Lysis, RT and PCR was performed as previously described (Islam et al., Nat Methods. 2014 Feb;11(2):163-6). Amplified cDNA was harvested with 13 microlitre Harvest Reagent and cDNA library quality was measured on an Agilent BioAnalyzer. Cell barcoding and fragmentation was performed in a single step using Tn5 DNA transposase as described previously. 1 microlitre Dynabeads MyOne Streptavidin C1 beads (Invitrogen) were resuspended in 20 microlitre Binding and Blocking buffer (10mM Tris, 250mM NaCl, 5mM EDTA, 0.5% SDS) and added to each well. After 15 min incubation at room temperature, all wells were pooled, the beads washed once with 100 microlitre Washing buffer (10mM Tris-150mM NaCl, 0.02% Tween), once in 100 microlitre Qiagen Qiaquick PB and then twice using 100 microlitre Washing buffer. Restriction was performed to cleave 3’ fragments: the beads were incubated in 100 microlitre restriction mix (1x NEB CutSmart, 0.4 U/microlitre PvuI-HF enzyme) for 1h at 37°C. Finally, the beads were washed three times with Washing buffer, then resuspended in 30 μl ddH2O and incubated for 10 min at 70°C to elute the DNA. AMPure beads XP (Beckman Coulter) were used at 1.8x volume and eluted in 30 microlitre to remove short fragments.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Data processing |
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 dete cted at less than 4 molecules in the whole datasets were eliminated. Genome_build: UCSC hg19 Supplementary_files_format_and_content: Tab-delimited table of total number of detected mRNA molecules from each gene in each cell
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Submission date |
May 18, 2018 |
Last update date |
Dec 14, 2018 |
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 |
GPL11154 |
Series (1) |
GSE114670 |
Single cell RNA-seq of the human long-term self-renewing neuroepithelial-like stem cell line SAI2 |
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Relations |
BioSample |
SAMN09226044 |
SRA |
SRX4101427 |
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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