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Sample GSM7838342 Query DataSets for GSM7838342
Status Public on Oct 14, 2023
Title Mouse Colon, KO, rep 1 (KO1)
Sample type SRA
Source name Colon
Organism Mus musculus
Characteristics tissue: Colon
genotype: KO, Hnrnp Iflox/flox VillinCre/+
Extracted molecule total RNA
Extraction protocol Immediately after euthanasia and necropsy, whole colon tissues from WT and KO male mice (littermates, 3−month−old, male, n = 3) were washed twice with ice−cold PBS to remove fecal matter. Tissues were then opened by cutting longitudinally through the lu-men, and excess fat was removed during the process. Opened colon tissues were washed again in a clean petri dish containing ice−cold PBS and lightly swirled with forceps to re-move any remaining fecal matter. The cell isolation protocol was based on the manuscript published by Gracz et al. [1], with the modifications described below: • Epithelial cells: After washing, tissues were digested in ice−cold dissociation reagent #1 [47 mL PBS + 3 mL 0.5 M EDTA (Sigma, Livonia, MI, USA, #E9884) + 75 µL DTT (Sigma, #D0632)] for 20 min. Then, tissues were transferred to dissociation reagent #2 [47 mL PBS + 3 mL 0.5 M EDTA] for 10 min at 37 °C. After incubation, tissues with reagent were shaken for 30 s to release epithelium from the basement membrane, and remnant tissues consisting of submucosa and muscularis were removed for lamina propria digestion. The cell solution was centrifuged at 1000× g for 5 min at 4 °C to pellet the cells. Cell pellets were washed with 10 mL PBS containing 10% FBS (Sigma, Livonia, MI, USA, #F0926). The cell solution was then centrifuged again, and the pel-let was resuspended in 10 mL HBSS (Sigma, Livonia, MI, USA, #H6648) containing 8 mg Dispase (Sigma, Livonia, MI, USA, #D4693) and incubated for 10 min at 37 °C in a water bath. After incubation, the cell suspension was filtered through 70 and 40 µm nylon cell strainers (Falcon, #352350 and #352340) to exclude large clumps of cells. The filtered cell solution was pelleted again, washed with 10 mL HBSS containing 10% FBS, and then pelleted again. Finally, cells were resuspended in 1 mL RPMI 1640 (Corning Life Sciences, Corning, NY, USA, #15−040−CV) with 10% FBS. • Lamina propria cells: Remnant tissues from previous steps were transferred to diges-tion media (5 mL RPMI 1640 + 2 mg Dispase + 10 mg collagenase IV (Gibco, Grand Island, NY, USA, #17104−109) + 60 µL FBS) and cut into small pieces, incubated for 30 min at 37 °C with constant spinning. After incubation, the cell suspension was fil-tered through 70 and 40 um nylon cell strainers to exclude large clumps of cells, the same manufacturer as described before. The filtered cell suspension was pelleted again, washed with 10 mL HBSS containing 10% FBS, then pelleted again. Finally, cells were resuspended in 1 mL RPMI 1640 with 10% FBS.
[1] 21. Gracz, A.D.; Puthoff, B.J.; Magness, S.T. Identification, isolation, and culture of intestinal epithelial stem cells from murine intestine. Methods Mol. Biol. 2012, 879, 89–107.−1−61779−815−3_6.
Cellular suspensions (~5000 cells) were loaded on a Chromium Single−Cell Instru-ment (10× Genomics) to generate single−cell GEMs. Single−cell RNAseq libraries were prepared using Chromium Single−Cell Gene Expression NextGem V3.1 and sequenced on an S4 2 × 150 nt lane in a NovaSeq 6000 platform, following the manufacturer’s protocol (10× Genomics). Sequencing and library construction were performed at the Roy. J. Carver Biotechnology Center, University of Illinois at Urbana−Champaign.
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Data processing Reading files (150 nt in length) in FASTQ format were generated and de-multiplexed by Cell Ranger 3.1.0 (10× Genomics Cell Ranger 3.0.0) aligned with mm10 mouse reference genome.
The exonic reads uniquely mapped to the transcriptome were used for a unique mo-lecular identifier (UMI) counting. Subsequently, single cells and their UMI count matrices were imported into the R package “Seurat” (version 2.3.2) for further analysis.
Selection and filtering of the single−cell RNA sequencing data were carried out if the total UMI counts (cell counts) or the number of expressed genes (feature counts) of cells were beyond predefined thresholds, defined as the medians ± 3× the median absolute deviation (MAD).
We also discarded both the genes expressed in fewer than three cells and low−quality cells with ≤200 expressed genes. Furthermore, cells with >25% of the mitochondrial genes (markers for dead cells) were also discarded.
After the filtering process, as described above, a total of 31,798 cells collected from 6 animals were kept for subsequent analysis.
Assembly: mm10 mouse reference genome
Supplementary files format and content: The Rdata file contains the aggregated Seurat object with the following info in the metadata: 1) Orig.ident: the original identity of each Seurat object before merging; 2) nCount_RNA; 3)nFeature_RNA; 4) the percentage of mitochondrial RNA expressed in each cell; 5)nCount_SCT: the RNA counts after SCT transformation; 6)nFeature_SCT: the feature count after SCT transformation; 7)SCT_snn_res.0.5: cluster assignments for cells based on the SCTransform-normalized data, using the shared nearest neighbor clustering method at a resolution of 0.5.; 8)seurat_clusters; 9)genotype: WT or KO; 10) celltype: assigned manually based on cluster marker genes.
Supplementary files format and content: See above.
Submission date Oct 12, 2023
Last update date Oct 14, 2023
Contact name Guanying Bianca Xu
Organization name University of Illinois at Urbana-Champaign
Department Food Science and Human Nutrition
Lab Dr. Hong Chen's NEG Lab
Street address 905 S Goodwin Ave Rm #472B1
City Urbana
State/province Illinois
ZIP/Postal code 61801
Country USA
Platform ID GPL24247
Series (1)
GSE245188 Single-cell RNA Sequencing (scRNA−seq) Identifies L1CAM as a Key Mediator between Epithelial Tuft Cell and Innate Lymphoid Cell in the Colon of Hnrnp I Knockout Mice
BioSample SAMN37799412
SRA SRX22080230

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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