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| Status |
Public on Jun 29, 2024 |
| Title |
LOS_A3_S178_Aligned.quant |
| Sample type |
SRA |
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| Source name |
skin
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| Organism |
Mus musculus |
| Characteristics |
tissue: skin
cell type: hair follicle epithelial cell
genotype: wild type
hair follicle_epithelia_region: lower outer root sheath
hair cycle_stage: AnaVI
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cells from 3-6 animals were pooled prior to FACS-isolation. In brief, cells were sorted into hypotonic lysis buffer, snap frozen in liquid nitrogen and stored at -80oC until all samples were collected. Cells were lysed by heating at 72oC for 3 min, followed by reverse transcription of mRNA using dT30 oligos, template switching oligos and Maxima H- reverse transcriptase. The whole transcriptome was amplified (15 cycles) by KAPA HiFi DNA polymerase (Roche), and then size-selected using 0.6X AmpPure XP beads (Beckman Coulter). To exclude cells with poor amplification, and wells containing multiple cells, qPCR for Gapdh was performed.
Illumina sequencing libraries were indexed with unique 5’ and 3’ barcode combinations (up to 384 cells) using the Nextera XT DNA library preparation kit (Illumina). Libraries were pooled and size-selected with 0.9X AmpPure XP beads. Prior to sequencing on Illumina NextSeq500 using a 75 bp paired-end read mid-output setting, library quality was assessed by TapeStation (Agilent).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
raw sequencing reads were aligned to the mouse reference genome (UCSC release mm39) using STAR (v2.6)
The expression values of each gene were quantified as both raw counts and transcripts per million (TPM) using Salmon (v.1.4.0), and compiled in R (v.3.6.1) using RStudio (v.3.4.2) by Tximport (v.1.12.3)
Raw count and metadata matrices for HFSCs across the hair cycle were loaded in SCANPY as an AnnData object. Single cell data was preprocessed to remove lowly detected genes (expressed in <75 cells) and cells with low complexity libraries (<2,000 genes detected).
SCANPY was used to normalize counts per cell, and highly variable genes were detected. Prior to dimensionality reduction by principal component analysis (PCA), data was centered and scaled. PCA was performed on highly variable genes, with 100 components and the svd_solver using ‘arpack’ (SCANPY default setting). To construct a k-nearest neighbours graph on Euclidean distance, 41 principal components were used (which captured 25% of the variance in the data). Data was visualized using uniform manifiold projection (UMAP) in SCANPY, and clustering was done using the Leiden algorithm (with a resolution setting of 0.5). Cluster resolution was chosen after iterating through resolution parameters from 0.1 to 0.75, as best capturing both hair follicle cycle stages and anatomic location (upper bulge region/upper ORS versus hair germ/upper-middle ORS versus lower ORS).
Assembly: mm39
Supplementary files format and content: raw count matrix as txt
Supplementary files format and content: metadata for samples as txt
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| Submission date |
Jun 29, 2024 |
| Last update date |
Jun 29, 2024 |
| Contact name |
Katherine Stewart |
| Organization name |
The Rockefeller University
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| Department |
Laboratory of Mammalian Cell Biology and Development
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| Lab |
Elaine Fuchs
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| Street address |
1230 York Avenue
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| City |
New York City |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE271007 |
Stem cells tightly regulate dead cell clearance to maintain tissue fitness |
| GSE271100 |
Stem cells tightly regulate dead cell clearance to maintain tissue fitness [scRNA-seq] |
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| Relations |
| BioSample |
SAMN42176818 |
| SRA |
SRX25156163 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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