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Sample GSM5251045

Query DataSets for GSM5251045

Status

Public on Jan 10, 2022

Title

M2_Proximal_H2O

Sample type

SRA

Source name

Mouse proximal colon

Organism

Mus musculus

Characteristics

strain: C57/BL6
age: 12 weeks
tissue site: proximal colon
treatment: Water
Sex: female
mouse: M2

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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

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

Submission date

Apr 16, 2021

Last update date

Jan 10, 2022

Contact name

Alok Jaiswal

E-mail(s)

ajaiswal@broadinstitute.org

Organization name

The Broad Institute of MIT and Harvard

Street address

415 Main Street