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| Status |
Public on Apr 03, 2023 |
| Title |
AOM/DSS lesion2 |
| Sample type |
SRA |
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| Source name |
Distal colon
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| Organism |
Mus musculus |
| Characteristics |
cell type: Microdissected Lesion
tissue: Distal colon
strain: C57BL/6
age: 23 weeks
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| Extracted molecule |
total RNA |
| Extraction protocol |
Isolated intestines from euthanized mice were washed with ice-cold PBS; villi were scraped using glass slides and dissociated in 5mM EDTA in PBS at 4ºC for 20 minutes, shaking every 5-7 minutes for about 30 seconds. The epithelial fraction was collected by centrifugation and incubated at 37ºC for 30 minutes in pre-warmed 4x TrypLE. Single cells filtered through 70um filter post trypsinization, washed with complete media, and collected in FACS buffer.
About 17,000 viable cells were loaded onto a 10X Genomics ChromiumTM instrument (10× Genomics) according to the manufacturer’s recommendations. The scRNAseq libraries were processed using ChromiumTM single cell 5’ or 3' library & gel bead kit (10× Genomics). Matched cell hashing libraries were prepared using single cell 5’ feature barcode library kit. Quality controls for amplified cDNA libraries, cell hashing libraries, and final sequencing libraries were performed using Bioanalyzer High Sensitivity DNA Kit (Agilent). The sequencing libraries for scRNAseq and scTCRseq were normalized to 4nM concentration and pooled using a volume ratio of 4:1. The pooled sequencing libraries were sequenced on Illumina NovaSeq S4 300 cycle platform. The sequencing parameters were: Read 1 of 150bp, Read 2 of 150bp and Index 1 of 8bp. The sequencing data were demultiplexed and aligned to mm10 or hg19-3.0.0 using cell ranger version 3.1.0 pipeline (10× Genomics).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
10x Genomics
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| Data processing |
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v2.1.1 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Pre-processing, alignment and gene counts: De-multiplexing, alignment to the transcriptome, and unique molecular identifier (UMI) collapsing were performed using the Cellranger toolkit provided by 10X Genomics.
General Clustering: Standard procedures for QC filtering, data scaling and normalization, detection of highly variable genes, and hashtag oligo (HTO) demultiplexing were followed using the Seurat v3 in RStudio. Cells with unique feature counts lower than 100 and greater than 25,000 as well as cells with greater than 25% mitochondrial DNA were excluded. Counts were log-normalized and scaled by a factor of 10,000 according to the default parameters when using the Seurat LogNormalize function. Variable features were identified, and the data were scaled using the default parameters (Ngenes = 2000) of the FindVariableFeatures FIG. caleData Seurat functions, respectively. HTOs were demultiplexed using the HTODemux function, and cells were identified as containing HTO-1 or HTO-2 based on their maximal HTO-ID signal. The cell population was filtered to contain only HTO-positive, singlet cells for further analysis.
HASHING CONDITIONS:72 day induced samples: - HTO-1 = ApcKO, Sox9WT (mouse 2792) - HTO-2 = ApcKO, Sox9 F/+ (mouse 3012) 30 day induced sample: - HTO-3 = ApcKO, Sox9 F/+ (mouse 3047)
Note: The Day 28: WT and Sox9KO and Day 10 data were done using 5' kit with single index, while the rest of the samples were done with 5' v2 kit with dual index.
Assembly: mm10,hg19
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Feb 06, 2023 |
| Last update date |
Apr 03, 2023 |
| Contact name |
Nilay Sethi |
| E-mail(s) |
nilay_sethi@dfci.harvard.edu
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| Organization name |
DANA-FARBER CANCER INSTITUTE
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| Street address |
450 Brookline Ave
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| City |
BOSTON |
| State/province |
Massachusetts |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE221300 |
Aberrant cell state plasticity mediated by developmental reprogramming precedes colorectal cancer initiation |
| GSE224679 |
Aberrant cell state plasticity mediated by developmental reprogramming precedes colorectal cancer initiation [scRNA-seq] |
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| Relations |
| BioSample |
SAMN33143546 |
| SRA |
SRX19295174 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7029394_111_12_barcodes.tsv.gz |
48.6 Kb |
(ftp)(http) |
TSV |
| GSM7029394_111_12_features.tsv.gz |
284.1 Kb |
(ftp)(http) |
TSV |
| GSM7029394_111_12_matrix.mtx.gz |
40.5 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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