|
| Status |
Public on Feb 03, 2023 |
| Title |
Dermal cells, D0, scRNAseq |
| Sample type |
SRA |
| |
|
| Source name |
neonatal mouse derma
|
| Organism |
Mus musculus |
| Characteristics |
tissue: neonatal mouse derma
time: 0 days of transdifferentiation
|
| Treatment protocol |
A single-cell suspension from neonatal dermal was prepared by mechanical and enzymatic dissociation as previously described (Hou et al., 2013). In brief, the limbs and tail were removed from the euthanized new-born mice (1–3 d) before gently pulling away the skin from the body. The skin was digested with 0.35% collagenase II in a 37 °C water bath for 1hr.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
The single-cell suspension was then subjected to a BD Influx cell sorter to exclude debris and doublets. Cells were pelleted and washed with 3 times PBS with 0.04% BSA twice to remove ambient RNA as well as minimize cell aggregation. Cells were filtered using a 70-mm cell strainer to remove any remaining cell debris and large clumps.
Library was performed according to the manufacter’s instructions (single cell 3’ v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
10x Genomics
|
| Data processing |
Single cell RNA-seq data were aligned to mm10 genome reference and counted the expression of genes of each barcode using cellranger (V3.0.2)
We loaded filtered feature matrixes into R by package DropletUtils
Barcodes with less than median-3*median absolute deviation total UMI counts or barcodes with less than median-3*median absolute deviation total features or barcodes with higher than median+2*median absolute deviation proportion of total mitochondrial counts were defined as low quality cells which were removed for downstream analysis using R package scater.
Then we normalized UMI count matrixs by R package scran and imported into the Seurat (V3.2.3) R toolkit for downstream analysis.
Assembly: mm10
Supplementary files format and content: Tab-separated values files, comma-separated values and matrix files
|
| |
|
| Submission date |
Sep 12, 2022 |
| Last update date |
Feb 04, 2023 |
| Contact name |
Yaqi Wang |
| E-mail(s) |
yaqi_wang@pku.edu.cn
|
| Organization name |
Peking University
|
| Street address |
No.5 Yiheyuan Road Haidian District
|
| City |
Beijing |
| State/province |
Beijing |
| ZIP/Postal code |
100871 |
| Country |
China |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE213195 |
Chemical reprogramming of melanocytes to skeletal muscle cells |
|
| Relations |
| BioSample |
SAMN30813867 |
| SRA |
SRX17539012 |
