|Public on Jun 07, 2023
|2-day miR-205 inducible Rep2
|P44 hair follicle stem cells
|cells: hair follicle stem cells
treatment: treated with doxcyclin for 2 days
|miR-205 inducible mouse was feed Doxcyclin chow for 2 days from P42-P44 or for 4 days from P49-P53.
|Gender matched mice (Ctrl and miR-205Ind) harboring a K14-H2BGFPallele were fed doxycycline for 2 or 4 days and euthanized to collect back skin samples. Hair coats were shaved and removed. Subcutaneous fat was removed, and back skin samples were minced into small pieces and incubated with 0.25% collagenase (Worthington, LS004188) in 10 mL 1x HBSS buffer at 37°C for 2 hours with rotation. After collagenase treatment, 10 mL cold PBS was added, and sample suspension was centrifugated at 400 g for 10 minutes at 4°C to get the pellet. The pellet was resuspended with 5 ml pre-warmed 0.25% trypsin/EDTA (Gibco) for 7 minutes at 37°C and the digestion was blocked by adding 10 mL cold 1x PBS with 3% chelated FBS. The suspension was extensively triturated with a 25 ml pipette and filtered through a 40 m cell strainer, followed by centrifugation at 400 g for 5 minutes at 4°C. The pelleted cells were resuspended in cold 1x PBS with 3% chelated FBS. Suspended cells were incubated with appropriated antibodies for 1 hour on ice. After washing away unbounded antibodies, DAPI was used to exclude dead cells. Live hair follicle stem cells from both control and miR-205 inducible animals were collected from FACS sorting machine with cell surface proteins and H2BGFP signals.
Total RNAs from FACS-purified cells were isolated using TRIZOL (Invitrogen). RNA quality was assessed by Agilent 2100 bioanalyzer. RNA Samples with RNA integrity numbers (RIN) > 8 were used to make libraries and perform RNA-seq assay. Libraries were prepared by following manufacturer’s protocol (NEBNext Ultra Directional RNA Library Prep Kit). The cDNA libraries were quality-checked with bioanalyzer and sequenced at Genomics and Microarray Core Facility at University of Colorado Denver on Illumina NovaSeq 6000.
|Illumina NovaSeq 6000
|RNA-seq reads (150nt, paired-end) were aligned to the mouse genome (NCBI37/mm10) using Hisat2 (version 2.1.0).
The resulting sam files were converted to bam files using SAMtools (version 1.3.1).
Expression measurement of each gene was calculated from the resulting alignment bam file by HT-seq.
Differentially expressed genes were determined using DEseq2 using default settings.
Supplementary_files_format_and_content: tab-delimited text files include baseMean, log2FoldChange, pValue..
|Sep 30, 2021
|Last update date
|Jun 07, 2023
|Pathology, Feinberg School of Medicine
|765 N FAIRBANKS CT
|Transcriptome analysis in hair follicle stem cells after miR-205 induction in mouse
|RNA-seq for miR-205 inducible hair follicle stem cells and ScRNAseq for miR-205 inducible back skin samples