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Sample GSM7679762 Query DataSets for GSM7679762
Status Public on Sep 20, 2023
Title MISTRG6 scleroderma skin graft 6 [HSC-engrafted]
Sample type SRA
Source name Skin
Organisms Homo sapiens; Mus musculus
Characteristics tissue: Skin
treatment: HSC engrafted
Extracted molecule total RNA
Extraction protocol Skin grafts were incubated in RPMI 1640 medium (Gibco) containing 5% fetal bovine serum (5% FBS/RPMI) and 10 mg/ml Dispase II (Sigma D4693-1G) for 45 minutes at 37° C shaking at 200-250 rpm. The sample was then removed from the media and minced with sterile iris scissors, followed by digestion with Liberase TM (Sigma) 0.5 mg/ml and DNase I 30 Units/ml in 5% FBS/RPMI for 45 minutes at 37° C shaking at 200-250 rpm. The resulting single cell suspension was then filtered through a 70 mm nylon membrane and washed. Live cells were sorted on a FACSAria and their final concentration and viability quantified with Trypan blue on a hemacytomer. The cells were pelleted and suspended in phosphate buffered saline containing 0.04% bovine serum albumin between 500-1000 cells/ml. 3000-6000 cells with greater than 80% viability were submitted to the Yale DNA Sequencing facility for generation of single-cell cDNA libraries using the Chromium Single Cell Controller (10x Genomics).
per 10x Genomics protocol using Single Cell 3' v2. Each cDNA library was sequenced paired-end on 1 lane with 75 base-pair read length using the Illumina HiSeq 2500 System generating at least 75,000 reads per cell.
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina HiSeq 2500
Description 10x Genomics
Data processing The 10x genomics Cell Ranger pipeline was used to align the reads, perform clustering and gene expression analysis, and aggregate the samples with normalized read counts. All 10x experiments were completed with the same 3’ chemistry and high-throughput sequencer to avoid batch effects. CellRanger was run 3 times using 3 different genomes: mm10, hg19, and a combined mm10 and GRCh38 genome. The analysis for the mm10, hg19, and combined mm10-GRCh38 genome was conducted separately but followed the same procedure. The raw matrices from Cell Ranger each of the genomes were processed and analyzed using Seurat version 3 R toolkit for single cell genomics (76, 77). For the matrices aligned to the mm10 genome and the combined mm10-GRCh38 genome, low quality cells with a high mitochondrial percentage of over 7.5% were filtered out. For the matrices aligned to the hg19 genome, low quality cells with a mitochondrial percentage of over 10% were filtered out. After initial filtering, the data was normalized (log normalized using the default scaling factor of 10000), scaled, principal components were calculated, and the data was visualized using a UMAP embedding of the data. After determining the clusters, canonical markers were used to identify the different cell types present. Once the canonical markers were identified, we used DAseq (27) to identify the regions that were differentially abundant between HSC engraftment and unengrafted samples. From DAseq, we were able to identify cell types that had differentially abundant regions and then looked at differential gene expression in those cell types using Seurat. After differential expression analysis, we performed pathway analysis of genes with adjusted p-value of less than 0.05 using Metascape (78) to identify pathways associated with genes upregulated in either the HSC or unengrafted samples
Assembly: GRCh38 and mm10
Supplementary files format and content: h5
Submission date Aug 03, 2023
Last update date Sep 20, 2023
Contact name Ian Odell
Organization name Yale University
Street address 300 Cedar St. Tac-S570
City New Haven
State/province CT
ZIP/Postal code 06520
Country USA
Platform ID GPL22245
Series (1)
GSE240009 IL-6 Trans-Signaling in a Humanized Mouse Model of Scleroderma
BioSample SAMN36829934
SRA SRX21241710

Supplementary file Size Download File type/resource
GSM7679762_HSC_3_raw_feature_bc_matrix.h5 12.8 Mb (ftp)(http) H5
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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