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Sample GSM2835689

Query DataSets for GSM2835689

Status

Public on Dec 21, 2017

Title

GHA501A171, Untreated single cell RNA-seq

Sample type

SRA

Source name

Keratinocytes

Organism

Mus musculus

Characteristics

treatment: UnTreated - 24 hours 4OHT - Control
cell type: Keratinocytes

Treatment protocol

Untreated; DMSO. Treated; 200nM 4OHT for 24 hours.

Growth protocol

Keratinocytes were isolated and cultured from adult dorsal skin in FAD medium (one part Ham’s F12, three parts Dulbecco’s modified Eagle’s medium, 1.8×10-4 M adenine), supplemented with 10% foetal calf serum (FCS) and a cocktail of 0.5 μg/ml hydrocortisone, 5 μg/ml insulin, 1×10-10 M cholera enterotoxin and 10 ng/ml epidermal growth factor (HICE cocktail)

Extracted molecule

total RNA

Extraction protocol

Wild type and K14ΔNβ-cateninER keratinocytes were cultured on 12 well plates in a ratio of 9:1 for a total of 200,000 cells per well and allowed to attach for 24 hours. Subsequently, cells were treated with 4-OHT (200nM) or DMSO as a control. After 24 hours of treatment cells were trypsinised and resuspended as a single cell suspension.
Single keratinocytes were captured on a medium-sized (10-17μm) microfluidic chip (C1, Fluidigm). Cells were assessed for viability (LIVE/DEAD assay, Life Technologies) and C1 capture sites were imaged by phase contrast to determine empty and doublet capture sites. Cells were loaded onto the chip at a concentration of 300 cells μl-1. Doublet or non-viable cells were excluded from later analysis. Cell lysis, reverse transcription, and cDNA amplification were performed on the C1 Single-Cell Auto Prep IFC, as per the manufacturer’s instructions. For cDNA synthesis the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech) was used. Single cell Illumina NGS libraries were constructed with Nextera XT DNA Sample Prep kit (Illumina). Sequencing was performed on Illumina HiSeq4000 (Illumina) using 100bp paired-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

Single cell

Data processing

Reads were preprocessed using FastQC and Cutadapt. Sequences were aligned to the Mus Musculus genome (GRCm38) using Tophat discarding multiply-mapped reads. Gene level counts were extracted using featureCounts 53. Transcript levels were quantified as transcripts per million (TPM). Genes with a TPM greater than 1 were considered as expressed. We filtered cells for analyses on the basis of number of aligned reads (> 200,000), percentage of ribosomal reads (< 2%) and number of genes expressed (> 2000).
Genome_build: GRCm38
Supplementary_files_format_and_content: CSV containing TPM normalized read count for each gene in each cell

Submission date

Oct 27, 2017

Last update date

May 15, 2019

Contact name

Arsham Ghahramani

E-mail(s)

[email protected]

Organization name

Francis Crick Institute

Street address

1 Midland Road