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Status |
Public on Jan 10, 2022 |
Title |
M2_Proximal_DSS |
Sample type |
SRA |
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Source name |
Mouse proximal colon
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Organism |
Mus musculus |
Characteristics |
strain: C57/BL6 age: 12 weeks tissue site: proximal colon treatment: DSS Sex: female mouse: M2
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Extracted molecule |
total RNA |
Extraction protocol |
To obtain a stromal single cell suspension for single cell RNA-seq we collected colons from water- and chronic DSS-treated C57BL6J mice and flushed them with ice cold washing buffer (2% FBS in 1X PBS (Sigma-Aldrich, calcium and magnesium free)). After removing fat and any connective tissue, colons were flipped inside out using forceps and cut into 2 cm chunks. Each colon was then incubated with 25 mL epithelial strip buffer (5 mM EDTA (Invitrogen), 1 mM DTT (Sigma Aldrich), 2.5 mM HEPES (Gibco), and 5% FBS in HBSS (GE Healthcare, calcium and magnesium free)) for 30 minutes at 37ºC with stirring. Tissues were collected, rinsed with ice cold washing buffer, minced using scissors and razor blades, and transferred into 50 mL conicals containing 5 mL enzyme digest buffer (0.2 mg/mL Collagenase P (Roche), 0.2 mg/mL Dispase II (Gibco), 0.1 mg/mL DNase I (Roche) in full media (CO2 independent media (Gibco), 2% FBS, 1X GlutaMAX (Gibco), 1X MEM NEAA (Gibco))) and placed into a bead bath at 40ºC. The conicals were vortexed every 5 minutes for 10 minutes, tissue chunks were allowed to settle for 5 minutes, and the supernatants were collected into ice cold collection media (full media with 10% FBS and 10 mM EDTA) that was kept on ice. Next, 5 mL enzyme digest media was added to the remaining colon fragments, the conicals were vortexed every 5 minutes for 5 minutes, tissue chunks were allowed to settle for 5 minutes, and the supernatants were added to previously collected fraction. Then, 3 mL enzyme digest media was added to the remaining tissue fragments, pipetted up and down for 2 minutes, allowed to settle for 3 minutes, and supernatants were collected and added to the previously collected fraction; this process was repeated for ~45 minutes until no colon fragments remained. The collection buffer and its contents were filtered using a 100μm cell strainer (Falcon), centrifuged (10 minutes, 450g, 4ºC), and resuspended in full media (2% FBS, 10 mM EDTA). Lamina propria single cell suspensions obtained as above were blocked with FcR blocking reagent (Miltenyi Biotec) for 10 minutes on ice, stained with primary antibodies previously listed for 20 minutes on ice, and cells were sorted using the Beckman Coulter MoFlo Astrios EQ (100 μm nozzle, 25 psi). For all steps, cells were kept in full media (2% FBS, 10 mM EDTA). Stromal cells that were sorted were defined as Ter119-CD45-EpCAM-, and dead cells were excluded using NucBlue (1 drop/500 μL cells). Cells were collected in full media, and purity was assessed by taking a fraction of sorted cells and re-analyzing them immediately on the MoFlo Astrios EQ. All samples were analyzed using FlowJo (Tree Star). Single cell suspensions were processed using the Chromium Single Cell 3’ Gene Expression kit (v2, 10x Genomics) per manufacturer’s instructions. Libraries were sequenced on the Illumina instruments per manufacturer’s instructions.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Data processing |
Demultiplex and basecall to fastq with cellranger mkfastq pipeline made available by CellRanger v2.2 software (10x Genomics) FASTQ reads were aligned to the mm10 mouse transcriptome, to extract cell barcodes and UMI barcodes using the cellranger count function. A digital gene expression (DGE) matrix for each sample was processed further Cells that satisfied any one of the following criteria were removed: (1) < 300 detected genes; 2) outlier number of Unique Molecular Identifiers (UMIs); 3) outlier proportion of mitochondrial gene expression were excluded. Outlier cutoffs for each batch of samples were determined empirically based on the distribution of UMI and proportion of mitochondrial gene expression per cell; or 4) doublets identified by the python package Scrublet Genome_build: mm10 Supplementary_files_format_and_content: matrix.mtx: gene expression UMI count matrix of all intron and exon spanning transcripts are in sparse matrix format. Can be read using the Read10x function in Seurat R package barcodes.tsv: cell barcodes for each single cell corresponding to each column in the count matrix genes.tsv: Gene symbols corresponding to each row in the count matrix metadata.tsv: additional collected attributes for each cell barcode in count matrix. Contains informations on final annotated cell types, mouse sample, treatment, tissue site
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Submission date |
Apr 16, 2021 |
Last update date |
Jan 10, 2022 |
Contact name |
Alok Jaiswal |
E-mail(s) |
ajaiswal@broadinstitute.org
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Organization name |
The Broad Institute of MIT and Harvard
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Street address |
415 Main Street
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City |
Cambridge |
State/province |
MASSACHUSETTS |
ZIP/Postal code |
02142 |
Country |
USA |
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Platform ID |
GPL17021 |
Series (1) |
GSE172261 |
Single cell RNAseq of mouse colon stroma during chronic DSS and homeostatic condition |
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Relations |
BioSample |
SAMN18779595 |
SRA |
SRX10624841 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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