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| Status |
Public on May 07, 2024 |
| Title |
Colon, rockroi multimodal |
| Sample type |
SRA |
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| Source name |
Colon
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| Organism |
Mus musculus |
| Characteristics |
protocol: custom single-cell RNA-seq
tissue: Colon
cell type: Epithelial, Pdgfra positive fibroblasts
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| Extracted molecule |
total RNA |
| Extraction protocol |
Murine colonic tissues from Pdgfra-H2B-EGFP reporter mice (Hamilton et al., 2003) were flushed with PBS and finely minced into 2 mm pieces. Minced tissue fragments were washed three times with PBS. For the detachment of the epithelial fraction, the tissue pieces were incubated in Gentle Cell Dissociation Reagent (STEMCELL Technologies, Germany) while gently rocking for 30 minutes at room temperature. The pieces were pipetted up and down for the epithelial fraction to be detached. The epithelial fraction was filtered through a Falcon 70-μm cell strainer (Corning, Switzerland), washed with plain ADMEM/F12 and incubated for 5 minutes at 37°C in prewarmed TrypLE express (Gibco, Thermofisher, Switzerland). The gentleMACS Octo Dissociator (Miltenyi Biotec, Switzerland) m_intestine program was employed for single-cell dissociation. The obtained epithelial single-cell suspension was filtered through a Falcon 40-μm cell strainer (Corning) and kept on ice in ADMEM/F12 supplemented with 10% FBS. For dissociation of the mesenchymal fraction, the remaining tissue pieces (following epithelium detachment) were digested for 1 hour at 37°C under 110 revolutions per minute (rpm) shaking conditions in DMEM supplemented with 2 mg/mL collagenase D (Roche) and 0.4 mg/mL Dispase (Gibco). The mesenchymal fraction was filtered through a Falcon 70-μm cell strainer (Corning), washed with plain ADMEM/F12, and subsequently filtered through a Falcon 40-μm cell strainer (Corning). The epithelial and mesenchymal cells were mixed and stained for 30 minutes on ice with anti-CD326(EpCAM)-PE-Cy5 (1:500, eBioscience/Thermofisher, Switzerland) in PBS. Prior to cell sorting, all cells were stained for 5 minutes on ice with DAPI in PBS (1:1000, ThermoFisher, Switzerland). Epithelial and mesenchymal cells labeled with PE-Cy5 and EGFP were sorted separately and subsequently mixed in a 1:1 ratio. Cells were sorted at the Cytometry Facility at the University of Zürich using a FACSAria III cell sorter (BD Biosciences, Switzerland).
Libraries were generated on a BD Rhapsody Express machine. Libraries for unmodified samples were generated following manufacturers recommendations for the WTA assay. The protocol for v1 BD Rhapsody was used (i.e. a single RPE reaction) and applied to enhanced beads. The RoCK and ROI sample was generated following the RoCK and ROI protocol. Briefly, the protocol differs from the standard WTA protocol due to the following points: 1) addtion of N1 primer to the RPE PCR; 2) addition of ROIseq primers to the pool of randomeres during RPE extension; 3) separate indexing reaction for the samples in which the N1 primer is added; 4) custom sequencing primer
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
BD Rhapsody
custom_regions_pdgfra.gtf.gz
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| Data processing |
The mapping, barcode filtering and counting was performed using a custom Snakemake pipeline (https://github.com/imallona/rock_roi_method/) based on STARSolo and featureCounts. Count matrices were analysed in R.
Index the reference genome with STAR.
Subset reads matching the WTA cell barcodes and map those to the transcriptome (genome plus GTF) using STARsolo. Detected cell barcodes (cells) are filtered in at two levels: first, by matching to the user-provided cell barcode whitelist; and second, by applying the EmptyDrops algorithm to discard empty droplets. We report two outputs from this step: the filtered-in cells according to the aforementioned filters; and the unbiased, whole-transcriptome WTA count table
Subset reads matching both the TSO CB structure and the filtered in cell barcodes and map those to the transcriptome. Our reasoning is that the expected TSO transcriptional complexity is undefined and not usable to tell apart cells from empty droplets, so we borrow the filtered-in cells from the EmptyDrops results from the WTA analysis.
(optional) Count on-target features in a more lenient way, filtering in multioverlapping and multimapping reads. This run mode is recommended when the captured regions target non unique loci (i.e. repetitive sequences)
Assembly: Combined genome, mouse: GRCm38.p6.genome.fa and gencode.vM25.annotation.gtf. Additional sequences: eGFP, and custom sequences of mouse strain
Supplementary files format and content: STARsolo output files wta: tab-delimited tsv and sparse matrix mtx files; filtered by EmptyDrops barcodes (matrix.mtx, barcodes.tsv, features.tsv)
Supplementary files format and content: STARsolo output files tso: tab-delimited tsv and sparse matrix mtx files; raw counts (matrix.mtx, barcodes.tsv, features.tsv)
Supplementary files format and content: tab-delimited featureCounts table for wta for wta and tso (wta_featurecounts.tsv, tso_featurecounts.tsv)
Supplementary files format and content: Bigwig coverage track for wta and tso (tso.bw and wta.bw)
Supplementary files format and content: GTF file containing information on the custom RoCK and ROI regions (custom_regions_pdgfra.gtf)
Supplementary files format and content: comma-separated table containing information on the broad cell (mesenchymal vs epithelial) and cluster annotation for each cell barcode kept for analysis after doublet removal and QC filtering (annotation.csv)
Library strategy: custom scRNAseq
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| Submission date |
Apr 29, 2024 |
| Last update date |
May 07, 2024 |
| Contact name |
Giulia Arianna Moro |
| E-mail(s) |
giulia.moro2@uzh.ch
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| Organization name |
University of Zurich
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| Department |
DMLS
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| Lab |
Prof. Dr. Konrad Basler
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| Street address |
Winterthurerstrasse 190
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| City |
Zürich |
| ZIP/Postal code |
8057 |
| Country |
Switzerland |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE266160 |
RoCK and ROI: a single-cell RNA-sequencing method with enhanced transcriptome information via targeted enrichment of transcripts and preselected, region-specific sequencing III |
| GSE266161 |
RoCK and ROI: a single-cell RNA-sequencing method with enhanced transcriptome information via targeted enrichment of transcripts and preselected, region-specific sequencing |
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| Relations |
| BioSample |
SAMN41118359 |
| SRA |
SRX24401318 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8241647_colon_rockroi_multimodal_annotation.csv.gz |
36.3 Kb |
(ftp)(http) |
CSV |
| GSM8241647_colon_rockroi_multimodal_tso.bw |
41.9 Mb |
(ftp)(http) |
BW |
| GSM8241647_colon_rockroi_multimodal_tso_featurecounts.tsv.gz |
48.8 Kb |
(ftp)(http) |
TSV |
| GSM8241647_colon_rockroi_multimodal_tso_raw_barcodes.tsv.gz |
179.2 Mb |
(ftp)(http) |
TSV |
| GSM8241647_colon_rockroi_multimodal_tso_raw_features.tsv.gz |
455.3 Kb |
(ftp)(http) |
TSV |
| GSM8241647_colon_rockroi_multimodal_tso_raw_matrix.mtx.gz |
11.3 Mb |
(ftp)(http) |
MTX |
| GSM8241647_colon_rockroi_multimodal_wta.bw |
143.1 Mb |
(ftp)(http) |
BW |
| GSM8241647_colon_rockroi_multimodal_wta_featurecounts.tsv.gz |
50.3 Kb |
(ftp)(http) |
TSV |
| GSM8241647_colon_rockroi_multimodal_wta_filtered_barcodes.tsv.gz |
34.3 Kb |
(ftp)(http) |
TSV |
| GSM8241647_colon_rockroi_multimodal_wta_filtered_features.tsv.gz |
455.3 Kb |
(ftp)(http) |
TSV |
| GSM8241647_colon_rockroi_multimodal_wta_filtered_matrix.mtx.gz |
38.4 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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