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Sample GSM4105916 Query DataSets for GSM4105916
Status Public on Jul 07, 2022
Title Skin-derived fibroblasts Patient 2
Sample type SRA
 
Source name Skin-derived fibroblasts Patient 2
Organism Homo sapiens
Characteristics cell type: Skin-derived fibroblasts
library preparation: 10X Genomics Single Cell RNAseq v3
Growth protocol Skin tissue was washed and epithelium removed by incubating with dispase (5 mg/ml) for 1.5 hr at 37 °C. Nerve sample was washed and fascicles pulled out from Sciatic nerve. Then both samples went through identical steps which were then cut into small cubes and incubated with DMEM with 10% fetal bovine serum (FBS), forskolin (Fsk, 5 µM), neuregulin (NRG, 50 ng/ml) and plasmocin (0.02%) for 2 weeks. At the end of 2 weeks, tissue explants were digested overnight at 37 °C with enzymatic cocktail containing dispase (1.25 U/ml) and collagenase IV (0.125%). The cell suspension was filtered through 40 µm filter and seeded onto poly-D-lysine and laminin coated cell culture dish, supplemented with DMEM with 2% FBS, Fsk (5 µM) and NRG (50 ng/ml). After 1 week, SCs were enriched using immunopanning against primate specific p75 antibody and fibroblasts from skin selected against human specific Thy-1 antibody. Purified cells were expanded in DMEM with 2% FBS, Fsk (5 µM) and NRG (50 ng/ml).
Extracted molecule polyA RNA
Extraction protocol Dissociation from cell culture dish and resuspension in BSA 1%/HBSS
Libraries were prepared according to 10X Genomics ChromiumTM Single Cell 3’ Reagent Guidelines v2 for Patient 1 and v3 for Patients 2 and 3 Chemistry as per the manufacturer’s protocol. In brief, single cells were sorted into 1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR over 10 cycles. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. Sequencing was performed first using Illumina MiSeq SR50 to approximate the number of recovered cells in each sample.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description 6000 cells loaded
Data processing All raw FASTQs reads were aligned to the human referene genome GRCh38 (primary assembly) using the STAR algorithm and quantified through the 10X Genomics cellranger v3.0.0 single cell pipeline, with default and recommended parameter. Only mapped reads were used for normalization before aggregating cells. Since we aggregated libraries constructed with different 10X chemistry versions, cellranger aggr’s batch correction algorithm was used to correct for systematic variability in gene expression profiles due to different versions of the 10X chemistry. The resulting gene-barcode matrix was imported into Seurat (v3 ) R toolkit for quality control, cross-patient integration, and all downstream analysis. All functions were run with default or recommended parameters, unless specified otherwise. Low quality cells (<300 genes/cell and <20 cells/gene) were excluded from the overall experiment. Gene expression was log normalized to a scale factor of 10,000. To begin unsupervised clustering of cells, a selection of highly variable genes was obtained and used to calculate up to 50 principle components. Significance of principle components was determined using JackStraw analysis and by creating a PCElbowPlot. PCs 1 to 25 were used to run and plot a tSNE plot for all cells.
Genome_build: Homo_sapiens : GRCh38
Supplementary_files_format_and_content: Filtered gene - barcode matrix output from CellRanger
 
Submission date Oct 02, 2019
Last update date Jul 10, 2022
Contact name Jeff Biernaskie
E-mail(s) jabierna@ucalgary.ca
Phone 4032107306
Organization name University of Calgary
Department Comparative Biology and Experimental Medicine
Lab Biernaskie Lab
Street address 3330 Hospital Drive NW
City Calgary
State/province Alberta
ZIP/Postal code T2N4N1
Country Canada
 
Platform ID GPL24676
Series (1)
GSE125422 Comparison of human skin- and nerve-derived Schwann cells reveals many similarities and subtle genomic and functional differences
Relations
BioSample SAMN12900235
SRA SRX6941570

Supplementary file Size Download File type/resource
GSM4105916_sample_4_barcodes.tsv.gz 21.9 Kb (ftp)(http) TSV
GSM4105916_sample_4_features.tsv.gz 264.3 Kb (ftp)(http) TSV
GSM4105916_sample_4_matrix.mtx.gz 66.9 Mb (ftp)(http) MTX
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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