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Sample GSM1650644

Query DataSets for GSM1650644

Status

Public on Sep 20, 2016

Title

1772067075_C12

Sample type

SRA

Source name

epidermis

Organism

Mus musculus

Characteristics

cell type level 1: uHF-I
strain: C57BL/6
Sex: female
age: P56
tissue: epidermis

Growth protocol

C57BL/6 mice (Charles River) were sacrificed at P56 +/-4 days and hair cycle stages were determined by staining dorsal skin sections for Ki67. Mice that showed signs of early anagen were excluded from this analysis. All animal experiments were performed in accordance with the Swedish legislation.

Extracted molecule

total RNA

Extraction protocol

Clipped and disinfected dorsal skin was isolated, dermal and adipose tissue was removed and strips of skin were floated on trypsin (Sigma) for 2h at 32°C. Epidermal tissue was subsequently scraped into S-MEM / 1% BSA and single cells were isolated by magnet stirring at 120 rpm for 25 min / RT. The resulting cell suspension was filtered through 70 µm and 40 µm cell strainers, resuspended in in Defined Keratinocyte Serum-free Medium (DK-SFM) without supplement (Life Technologies) and Sca1+ and Sca1- were separated using Anti-Sca1-FITC magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions. Cells were stored on ice in DK-SFM with 0.1 mg/ml DNase I (Stem Cell Technologies) until capturing. Before capturing, the cell suspension was carefully resuspended and two times passed through a 20 µm cell strainer. Epidermal cells were captured on a medium microfluidic chip (designed for cells from 10 µm – 17 µm) using the Fluidigm C1 Autoprep System. 14 µl filtered cell suspension (about 750 cells / µl in DK-SFM with DNase I) was mixed with 6 µl C1 Suspension Reagent and 14 µl were loaded onto the chip. Single-cells were then captured for 30 min at 4°C using the “Cell Load (1772x/1773x)” script.
Lysis mix, RT mix and PCR mix (described in Islam et al., Nat Methods. 2014 Feb;11(2):163-6) were added to the chip. The plate was placed in the Fluidigm C1 instrument and the 'mRNA Seq: RT + Amp (1772x/1773x)' script was executed, and included lysis, reverse transcription and 21 cycles of PCR. The amplified cDNA was harvested in a total of 13 μL C1 Harvesting Reagent. Amplified cDNA was simultaneously fragmented and barcoded by tagmentation using Tn5 DNA transposase to transfer adaptors to the target DNA as described (Ibid.). 100 µl Dynabeads MyOne Streptavidin C1 beads (Invitrogen) were washed in 2× BWT (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween-20) and resuspended in 2 ml 2× BWT. Twenty microliters of beads were added to each well and incubated at room temperature for 15 min. All fractions were pooled, the beads were immobilized and the supernatant removed. The beads were resuspended in 100 μL TNT (20 mM Tris, pH 7.5, 50 mM NaCl, 0.02% Tween), washed in 100 μL Qiagen Qiaquick PB, and twice in 100 μL TNT. The beads were resuspended in 100 μL restriction mix (1× NEB CutSmart, 0.4 U/μL PvuI-HF enzyme), designed to cleave 3′ fragments carrying a PvuI recognition site. The mix was incubated for 1 h at 37 °C, then washed three times in TNT. Finally, to elute DNA, beads were resuspend in 30 µL ddH2O and incubated 10 minutes at 70°C. Beads were then immediately bound to magnet and the supernatant was collected. To remove short fragments, Ampure beads (Beckman Coulter) were used at 1.8× volume and eluted in 30 µL.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

SingleCellNo189

Data processing

Read processing was performed as described (Islam et al., Nat Methods. 2014 Feb;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.
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

Submission date

Apr 06, 2015

Last update date

May 15, 2019

Contact name

Sten Linnarsson

Organization name

Karolinska Institutet

Department

Medical Biochemistry and Biophysics

Lab

Molecular Neurobiology