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Sample GSM4403045 Query DataSets for GSM4403045
Status Public on May 18, 2020
Title scRNA-seq_MAL inbibitor
Sample type SRA
Source name dorsal skin interfollicular epidermal basal cells under stretching and under CCG203971 treatment
Organism Mus musculus
Characteristics strain: CD1
treatment: stretching and CCG203971
cell type: dorsal skin interfollicular epidermal basal cells
Treatment protocol For the stretching condition, mice were anesthetized (5% xylazine 10% ketamine in PBS) and the dorsal skin was disinfected with 10% Iso-Betadine (Meda Pharma), an incision was created in the most caudal part of the dorsal skin to minimize any tension in the wound as the expansion progressed and to maximise the distance from the access wound, to the location of the expander. A subcutaneous pocket was created with forceps and a 4ml Hemispher Self-inflating tissue expander (OsmedTM) was placed in the most rostral part under the dorsal skin in proximity of the neck. Stiches were used to close the subcutaneous pocket, to limit the hydrogel movement and to close the access incision. All analyses were performed in the area on the top of the dome of the hemisphere induced by the skin expander, since this region experiences the highest strainFor TPA treatments, TPA (200 μl of 0.02 mg/ml solution in acetone) was administered daily to shaved mouse back skin for 2 days. To inhibit MEK1/2 activity, mice were treated with Trametinib 2mg per kg body weight by daily oral gavage. To inhibit MAL activity, mice received drug treatment via IP injection daily using 100mg CCG203971 (Cayman Chemical) per kg body weight for 2 or 4 days as indicated in the figures.
Growth protocol Mouse colonies were maintained in a certified animal facility in accordance with the European guidelines.
Extracted molecule total RNA
Extraction protocol To separate epidermis from dermis, the samples were incubated in HBSS (Gibco) 0,25% trypsin (Gibco) at 4°C overnight. Epidermis was mechanical separated from the dermis with scalpel first and then by pipetting and trypsin was neutralized by adding DMEM medium (Gibco) supplemented with 2% Chelex Fetal Calf Serum (FCS). Samples were filtrated on 70 and 40µm filter (Falcon). Cells were incubated in 2% FCS/PBS with primary antibodies for 30 min on ice, protected from the light, with shaking every 10 min. Immunostaining for isolating basal IFE and infundibulum cells was performed using FITC-conugated anti-α6-integrin (clone GoH3; BD biosciences). CD34 staining was used to exclude bulge cells from the sorted cells (clone RAM34; BD Biosciences). Primary antibodies were washed with 2% FCS/PBS and cells incubated for 30 min in APC conjugated streptavidin (BD Biosciences), on ice, with shaking every 10 min. Living epidermal cells were gated by forward scatter, side scatter and negative staining for Hoechst dye. Basal cells from the interfollicular epidermis were targeted using CD34 negative and α6 integrin positive gating. Fluorescence-activated cell sorting analysis was performed using FACSAria I at high pressure (70 psi) and FACSDiva software (BD Biosciences). After sorting, 6000 cells were loaded onto each channel of the Chromium Single Cell 3’ microfluidic chips (V2-chemistry, PN-120232, 10X Genomics) and individually barcoded with a 10X Chromium controller according to the manufacturer’s recommendations (10X Genomics). RNA from the barcoded cells was reverse transcribed, followed by amplification, shearing 5′ adaptor and sample index attachment.
The libraries were prepared using the Chromium Single Cell 3’ Library Kit (V2-chemistry, PN-120233, 10X Genomics), quantified using a low coverage Illumina NextSeq 550 run and sequenced on an Illumina NovaSeq
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Description D2_SRFinh
Data processing Read Alignment - CellRanger (version 3.0.2)
Cell-cycle assignment - Scran (1.10.1)
UMAP dimensionality reduction - Seurat (3.0.1)
Normalization - Seurat (3.0.1)
Gene regulatory network analysis - SCENIC (0.9.9+2.gcaded79)
Genome_build: mm10-1.2.0
Supplementary_files_format_and_content: R Rds objects
Submission date Mar 09, 2020
Last update date May 19, 2020
Contact name Cedric Blanpain
Organization name Université Libre de Bruxelles (ULB)
Lab Laboratory of stem cells and cancer
Street address 808, route de Lennik, CP 637
City Bruxelles
ZIP/Postal code 1070
Country Belgium
Platform ID GPL24247
Series (1)
GSE146637 Single Cell RNA-sequencing of murine back skin interfollicular epidermis basal cells
BioSample SAMN14337090
SRA SRX7878386

Supplementary file Size Download File type/resource
GSM4403045_ExpD2_SRFinh-Final-Seurat_v3.rds.gz 229.8 Mb (ftp)(http) RDS
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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