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| Status |
Public on Aug 25, 2020 |
| Title |
CD3I |
| Sample type |
SRA |
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| Source name |
stromal vascular cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: stromal vascular cells
technology: Bio-Rad SureCell
analysis_pipeline: bcbio
condition: Crohn's disease
patient_id: CD3
tissue_status: inflamed
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Adipose tissue was minced into 3 mm pieces and subjected to collagenase digestion for 50 min at 37°C with continuous rotation. Collagenase buffer consisted of 1X PBS containing calcium and magnesium, 2% bovine serum albumin (BSA; MP Bio, USA), 0.2 mg/mL DNase I (Sigma Aldrich, USA), and 1mg/mL collagenase II (Invitrogen, USA). Following digestion, cells were incubated with 0.01 M EDTA for an additional 10 min then filtered through a 100 µM cell strainer (Fisherbrand, USA) and centrifuged at 1500 rpm for 5 min. The pellet was resuspended in 1X RBC Lysis Buffer (Invitrogen, USA) as per manufacturer’s instruction. The samples were centrifuged as above, and the final pellet was converted to a single-cell suspension and frozen for later analysis such that all samples could be run at the same time. The cell-freezing protocol for scRNAseq was developed in conjunction with Bio-Rad Genomics. Briefly, the cell pellet was thoroughly mixed by pipetting up and down 10 times. Cells were counted a total of 4 times for each cell preparation to ensure accuracy of total cell count and viability. Aliquots of 5*10^6 cells/ml were prepared in chilled cryopreservation medium (DMEM + 20% FBS + 10% DMSO) and placed in a 4°C pre-chilled CoolCell FTS30 and placed in a -80C freezer for at least 4 hrs. After 4 hrs the cryovials were transferred to liquid nitrogen for long-term storage.
CrF vs. H-MAT. Single-cell libraries of the dissociated SVCs were prepared using the Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3 (10X Genomics) and the Chromium Chip B Single Cell Kit protocols, aiming for recovery of 1200 cells for each sample. Briefly, cells were individually partitioned and encapsulated into subnanoliter oil droplets in the Chromium Controller instrument for cell lysis and barcoded reverse transcription of mRNA, followed by amplification, shearing and Illumina library construction. Single-cell-barcoded cDNA libraries were sequenced on the Illumina NextSeq platform. CrF vs. CD MAT and UC MAT. Dissociated SVCs were first diluted to 2500 cells/uL in PBS with 0.1% BSA. Cells were individually partitioned and co-encapsulated with barcodes into subnanoliter oil droplets using the ddSEQ single-cell isolator (Bio-Rad, USA) as per manufacturer’s instructions in the Illumina Bio-Rad SureCell WTA 3' Library Prep Kit. Following cell isolation, the droplets were transferred to a 96-well PCR plate for cell lysis, barcoding, reverse transcription using a thermal cycler. The droplet emulsion was then disrupted for generation of second strand cDNA, followed by fragmentation, tagging and amplification of cDNA. Single-cell-barcoded cDNA libraries were sequenced on the Illumina NextSeq platform at an average read depth of 175,000 reads/sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
surecell_02
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| Data processing |
10X Genomics Cell Ranger sample processing. Cell counts were generated using the 10X Genomics Cell Ranger v3.1.0 pipeline against the GRCh38 reference genome (10X genome version 3.0.0; Ensembl release 93, July 2018; http://jul2018.archive.ensembl.org). The filtered count matrices generated by Cell Ranger were used as input. The Chromium R package v0.1.5 (https://chromium.acidgenomics.com) was used to perform additional cell quality control analysis and filtering prior to clustering. Similar to the approach used for the SureCell samples, UMIs per cell, genes per cell, and mitochondrial ratio cutoffs were applied to remove additional low quality cells from analysis.
Bio-Rad SureCell sample processing. Cell counts were generated with the bcbio-nextgen Python toolkit v1.1.1a0-06f3c2a9 (https://bcbio-nextgen.readthedocs.io). Reads were assigned per cell via the cellular barcodes, and per gene via the UMIs, using the umis toolkit v1.0.0 [@Svensson2017-hp]. Reads were quasi-mapped to the Ensembl GRCh38 transcriptome (Release 90, August 2017; https://aug2017.archive.ensembl.org) using Rapmap v0.5.0 [@Srivastava2016-tm]. Only cells containing at least 1,000 reads were analyzed. The bcbioSingleCell R package v0.4.12 (https://bioinformatics.sph.harvard.edu/bcbioSingleCell) was used to perform cell quality control analysis and filtering prior to clustering. The distributions of reads per cell, UMIs per cell, genes per cell, and mitochondrial ratio per cell were used to remove low quality cells from analysis.
Clustering analysis. Clustering analysis was performed with the Seurat R package v3.1.5. [@Satija2015-rr]. Counts were log normalized and scaled per cell to account for variations in sequencing depth. Linear dimensional reduction was performed using PCA on the most variable genes detected; these are determined via binned z-scores based on the average expression and dispersion for each gene (Satija et al., 2015). Non-linear dimensional reduction was performed using t-SNE [@Maaten2008-qy] and UMAP [@McInnes2018-aa, @Becht2018-jn]. We defined cell cluster specific marker genes using the Findmarkers function in Seurat across all samples using a Wilcoxon rank sum test. Differential expression analysis was performed using the edgeR package v3.30.3 [@Robinson2010-eu]. Plots were generated using the acidplots v0.2.29 (https://acidplots.acidgenomics.com/) and pointillism v0.4.11 (https://pointillism.acidgenomics.com/) R packages.
Genome_build: SureCell: Homo sapiens GRCh38 (Ensembl release 90); Chromium: Homo sapiens GRCh38 (10X genome version 3.0.0; Ensembl release 93)
Supplementary_files_format_and_content: Matrix Market Exchange Format (sparse matrix)
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| Submission date |
Aug 24, 2020 |
| Last update date |
Aug 25, 2020 |
| Contact name |
Michael J Steinbaugh |
| E-mail(s) |
mike@steinbaugh.com
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| Organization name |
Acid Genomics
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| Street address |
PO Box 1520
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| City |
Quechee |
| State/province |
VT |
| ZIP/Postal code |
05059 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE156776 |
Translocation of Viable Gut Microbiota to Mesenteric Adipose Drives Formation of Creeping Fat in Humans |
| GSE157026 |
Transcriptomics analysis of mesenteric adipose tissue from Crohn's disease patients and healthy controls |
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| Relations |
| BioSample |
SAMN15894769 |
| SRA |
SRX8999164 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4743743_CD3I.mtx.colnames.txt.gz |
92.9 Kb |
(ftp)(http) |
TXT |
| GSM4743743_CD3I.mtx.gz |
8.5 Mb |
(ftp)(http) |
MTX |
| GSM4743743_CD3I.mtx.rownames.txt.gz |
87.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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