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| Status |
Public on May 10, 2019 |
| Title |
HC-3 |
| Sample type |
SRA |
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| Source name |
Skin tissue
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| Organism |
Homo sapiens |
| Characteristics |
disease state: Healthy control
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Skin biopsies were minced and digested enzymatically (Whole Dissociation Skin Kit, Miltenyi Biotec) for 2 hours at 37°C and further dispersed using the Miltenyi gentleMACS Octo Dissociator. Experimental procedures followed established techniques7 using the Chromium Single Cell 3’ Library V2 kit (10x Genomics). Cell suspensions were separated by the Chromium System (10X Genomics)7,8 into mini-reaction "partitions" or GEMs formed by oil micro-droplets, each containing a gel bead and a cell. A 1000-fold excess of partitions compared to cells assured that most partitions/GEMs had only one cell per GEM. Gel beads contained an oligonucleotide scaffold composed of an oligo-dT section for priming reverse transcription, and barcodes for each cell (10X Genomics) and each transcript (unique molecular identifier, UMI), as described.9 7,000 cells were loaded into the instrument to obtain data on ~4,000 cells with a rate of ~3.1% of partitions showing more than one cell/partition. The following steps were all performed using reagents and protocols developed by 10X Genomics.
The emulsions were transferred from the Chromium chip to a PCR cycler for cDNA synthesis. The emulsion was then broken using a recovery agent, and following Dynabead and SPRI clean up, cDNAs were amplified by PCR (C1000, Bio-Rad). cDNAs were sheared enzymatically into lengths of ~200bp. DNA fragment ends were repaired, A-tailed, and adaptors ligated.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Chromium scRNA-seq data produced by the 10X Chromium Platform were processed to generate sample-specific fastq files.
Processed reads were then examined by quality metrics, mapped to a reference human genome using RNA-seq aligner STAR and assigned to individual cells of origin according to the cell specific barcodes, using the Cell Ranger pipeline (10X Genomics).
To ensure that PCR amplified transcripts were counted only once, only single UMIs were counted for gene expression level 10. In this way, cell x gene UMI counting matrices were generated for downstream analyses.
Seurat, an R package developed for single-cell analysis,11 was used to identify distinct cell populations and visualize cell clusters in graphs as in. 8 Specifically, the UMI matrix was filtered such that only cells expressing at least 200 genes were utilized in downstream analysis.
Unwanted sources of variation were regressed out of the data by constructing linear models to predict gene expression based on the number of UMIs per cell as well as the percentage of mitochondrial gene content.
Genome_build: GrCh38 genome
Supplementary_files_format_and_content: Raw dataframe csv files
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| Submission date |
Mar 19, 2019 |
| Last update date |
Jun 14, 2022 |
| Contact name |
Patrizia Fuschiotti |
| E-mail(s) |
paf23@pitt.edu
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| Organization name |
University of Pittsburgh
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| Street address |
200 Lothrop St
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| City |
Pittsburgh |
| State/province |
PA |
| ZIP/Postal code |
15261 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE128531 |
Single-cell lymphocyte heterogeneity inadvanced Cutaneous T-Cell Lymphoma skin tumors |
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| Relations |
| Alternative to |
GSM4450728 |
| Reanalyzed by |
GSM6243640 |
| BioSample |
SAMN11166219 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3679040_Labeled_SC124_080317_SK_NOR_GRCh38raw.csv.gz |
9.6 Mb |
(ftp)(http) |
CSV |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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