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| Status |
Public on May 07, 2024 |
| Title |
unmod_roi, second mixing experiment |
| Sample type |
SRA |
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| Source name |
Mix of L-cells and HEK293T cells
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| Organism |
Homo sapiens |
| Characteristics |
protocol: custom single-cell RNA-seq
cell line: Mix of L-cells and HEK293T cells
cell type: Mouse adult adipose, human embryonic kidney
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| Growth protocol |
HEK193 cells and L-cells cells were cultured in DMEM 1X with 10% FBS and 1% PIS in a 10 cm dish and maintained in an incubator at 37°C and 5% CO2
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were dissociated using Trypsin-EDTA, filtered through a cell strainer and resuspended in BD Rhapsody Sample Buffer
Libraries were generated on a BD Rhapsody Express machine. Libraries for unmodified samples (unmod) 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, either with prior RoCKseq modification (rock, rockroi conditions) or without modification (unmod, unmod_roi). rock, unmod_roi and rockroi conditions were 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 (for unmod_roi, rock and rockroi conditions); 2) addition of ROIseq primers to the pool of randomeres during RPE extension (for unmod_roi and rockroi conditions); 3) separate indexing reaction for the samples in which the N1 primer is added; 4) custom sequencing primer
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
BD Rhapsody
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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.tsv.gz. 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, human: GRCh38.p13.genome.fa and gencode.v38.basic.annotation.gtf. Additional sequences: eGFP and tdTomato
Supplementary files format and content: STARsolo output files wta: tab-delimited tsv.gz and sparse matrix mtx.gz files; filtered by EmptyDrops barcodes (matrix.mtx.gz, barcodes.tsv.gz, features.tsv.gz)
Supplementary files format and content: STARsolo output files tso: tab-delimited tsv.gz and sparse matrix mtx.gz files; raw counts (matrix.mtx.gz, barcodes.tsv.gz, features.tsv.gz)
Supplementary files format and content: tab-delimited featurecounts.tsv.gz table for wta for wta and tso (wta_featurecounts.tsv.gz.tsv.gz, tso_featurecounts.tsv.gz.tsv.gz)
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_mixing.gtf)
Supplementary files format and content: comma-separated table containing information on the species assignment for each cell barcode kept for analysis after doublet removal and QC filtering (species.csv)
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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 |
GPL24676 |
| Series (2) |
| GSE266117 |
RoCK and ROI: a single-cell RNA-sequencing method with enhanced transcriptome information via targeted enrichment of transcripts and preselected, region-specific sequencing II |
| 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 |
SAMN41113758 |
| SRA |
SRX24393397 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8240934_second_mixing_experiment_species_unmod_roi.csv.gz |
49.1 Kb |
(ftp)(http) |
CSV |
| GSM8240934_second_mixing_experiment_unmod_roi_tso.bw |
112.0 Mb |
(ftp)(http) |
BW |
| GSM8240934_second_mixing_experiment_unmod_roi_tso_featurecounts.tsv.gz |
53.5 Kb |
(ftp)(http) |
TSV |
| GSM8240934_second_mixing_experiment_unmod_roi_tso_raw_barcodes.tsv.gz |
2.6 Mb |
(ftp)(http) |
TSV |
| GSM8240934_second_mixing_experiment_unmod_roi_tso_raw_features.tsv.gz |
996.3 Kb |
(ftp)(http) |
TSV |
| GSM8240934_second_mixing_experiment_unmod_roi_tso_raw_matrix.mtx.gz |
31.8 Mb |
(ftp)(http) |
MTX |
| GSM8240934_second_mixing_experiment_unmod_roi_wta.bw |
169.7 Mb |
(ftp)(http) |
BW |
| GSM8240934_second_mixing_experiment_unmod_roi_wta_featurecounts.tsv.gz |
56.1 Kb |
(ftp)(http) |
TSV |
| GSM8240934_second_mixing_experiment_unmod_roi_wta_filtered_barcodes.tsv.gz |
35.3 Kb |
(ftp)(http) |
TSV |
| GSM8240934_second_mixing_experiment_unmod_roi_wta_filtered_features.tsv.gz |
996.4 Kb |
(ftp)(http) |
TSV |
| GSM8240934_second_mixing_experiment_unmod_roi_wta_filtered_matrix.mtx.gz |
54.5 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
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