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Sample GSM3146940

Query DataSets for GSM3146940

Status

Public on Dec 14, 2018

Title

C1-1772117-118_E09

Sample type

SRA

Source name

LT-NES SAI2 cell

Organism

Homo sapiens

Characteristics

cell type: long-term self-renewing neuroepithelial-like stem cells

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).

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

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

Submission date

May 18, 2018

Last update date

Dec 14, 2018

Contact name

Sten Linnarsson

Organization name

Karolinska Institutet

Department

Medical Biochemistry and Biophysics

Lab

Molecular Neurobiology