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Status |
Public on Sep 20, 2023 |
Title |
MISTRG6 scleroderma skin graft 1 [HSC-unengrafted] |
Sample type |
SRA |
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Source name |
Skin
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Organisms |
Homo sapiens; Mus musculus |
Characteristics |
tissue: Skin treatment: no HSC
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic single cell |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Description |
10x Genomics
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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
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Submission date |
Aug 03, 2023 |
Last update date |
Sep 20, 2023 |
Contact name |
Ian Odell |
E-mail(s) |
ian.odell@yale.edu
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Organization name |
Yale University
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Street address |
300 Cedar St. Tac-S570
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City |
New Haven |
State/province |
CT |
ZIP/Postal code |
06520 |
Country |
USA |
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Platform ID |
GPL22245 |
Series (1) |
GSE240009 |
IL-6 Trans-Signaling in a Humanized Mouse Model of Scleroderma |
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Relations |
BioSample |
SAMN36829939 |
SRA |
SRX21241705 |
Supplementary file |
Size |
Download |
File type/resource |
GSM7679757_noHSC_1_raw_feature_bc_matrix.h5 |
19.5 Mb |
(ftp)(http) |
H5 |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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