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Sample GSM8012754

Query DataSets for GSM8012754

Status

Public on Feb 21, 2024

Title

RNA-seq from skin keratinocytes 14 days following S.epi treatment in Ramp1 WT mice (rep3)

Sample type

SRA

Source name

skin

Organism

Mus musculus

Characteristics

tissue: skin
cell type: keratinocytes
genotype: Ramp1 WT
treatment: Staphylococcus epidermidis

Treatment protocol

Mice were treated with S. epidermidis every other day with 4 topical applications in total. Cells were harvsted 2 weeks after the first topical application

Extracted molecule

total RNA

Extraction protocol

Tissues were harvested and cells were extracted (see manuscript for details) and sorted. T cells were sorted as CD4+ CD25- CCR6- for Th1 cells and CD4+ CD25- CCR6+ for Th17. Keratinocytes were sorted as LD-Cd45-Cd31-Cd34-Cd49f+Sca1+. For more details for the protocol used to sort T cells refer to Linehan et al, 2018; For more details for the protocol used to sort keratinicytes refer to Lima-Junior et al., 2021
For T cells, cells were processed and analyzed for RNA-seq as described in Linehan et al., 2018. For keratiocytes: Keratinocytes from Lck-Cre Ramp1 fl/fl and WT mice were sorted as described in Lima-Junior et al., 2021.
For T cells: mRNA libraries were constructed using SMARTer Ultra Low Input RNA kit (Clonetech) + Nextera XT DNA library prep kit (Illumina). For keratinocytes: RNA libraries were construted using the mouse Tecan SoLo library preparation kit.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Data processing

For T cells, cells were processed and analyzed for RNA-seq as described in Linehan et al., 2018.
For keratinocytes: Sequencing reads were mapped to the mm10 (GRCm38) mouse genome using STAR with stringent mapping conditions, where reads aligned to multiple regions of the genome were eliminated (-outFilterMultimapNmax 2). Differential gene expression was calculated using HOMER’s getDiffExpression (Heinz et al. 2010). Genes with FDR < 0.05 and foldchange > 2 were considered differentially expressed transcripts. Retroelement and noncoding transcript expression was determined using HOMER, defining these transcripts based on the annotations provided by the Genome-based Endogenous Viral Element Database (gEVE) for GRCm38. For the analysis of individual loci, reads were analyzed using HOMER’s analyzeRepeats function (Heinz et al. 2010), along with a custom GFT file with the gEVE annotations. For the analysis of retroelement and noncoding transcript family motifs, reads were analyzed using the analyzeRepeats function with parameters “repeats mm10”.
Assembly: mm10 (GRCm38)
Supplementary files format and content: file format: txt
Supplementary files format and content: content for T cells: mean, foldchange and p-values; content for keratinocytes: TPM per replicate

Submission date

Jan 11, 2024

Last update date

Feb 21, 2024

Contact name

Verena Link

E-mail(s)

verena.link@nih.gov

Organization name

NIH

Department

NIAID