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| Status |
Public on Jul 17, 2024 |
| Title |
Sample_10_WTA |
| Sample type |
SRA |
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| Source name |
skin
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| Organism |
Mus musculus |
| Characteristics |
tissue: mouse epidermis
cell type: skin cells
strain: JAX #026179, CD1 wildtype x Tg(B6J.129(Cg)-Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J
age: 4 days post natal
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
P4 back skin was surgically removed and scraped for fat, the back skin was washed once in cold PBS and then placed in dispase (Corning; 354235) for 35 min at 37°C, epidermis facing up, on an orbital shaker. Next, epidermis was separated from dermis with fine forceps, torn into smaller pieces and placed in 4 mL 0.25% Trypsin-EDTA (1X, Gibco; 25200056) for 15 min at 37°C with orbital shaking. Epidermis was washed with cold PBS and pipetted vigorously to achieve a single cell suspension. Suspension was then afterwards filtered through a 70 µm and a 40 µm strainer (Corning; 431750, 431751) consecutively, then centrifuged for 10 min at 1400rpm and resuspended in FACS buffer (PBS + 2 % FBS(-) + DAPI) before sorting mCherry positive cells on a BD Aria II.
mCherry-positive cells were sorted on a BD FACSAria using a 70µm nozzle and processed for single-cell capture with a BD Rhapsody Single-Cell Analysis System. For P4 we loaded 8 separate cartridges (Cat No 633733) with an average of 50037 cells. Reverse transcription from beads and sequencing library production was carried out according to manufacturer's instructions (BD Biosciences, Doc ID: 210967 Rev. 1.0). Whole Transcriptome Amplification Analysis (WTAs) from BD Rhapsody Cartridges were sequenced in full SP flowcells at 1.8nM concentration with 20% PhiX and the following read configuration: Read1 60, I1 8, Read2 62 cycles.
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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 |
scRNA-seq WTA of P4 skin, mCherry CROP-seq, replicate 7. Same underlying biological sample as for Sample_10_guides
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| Data processing |
The raw sequencing data comprising the BCL files were demultiplexed using the Illumina bcl2fastq program v2.20 with default options allowing one mismatch of the sample barcode sequences. The resulting FASTQ files were processed using the BD Rhapsody™ Whole Transcriptome Analysis (WTA) Pipeline (v1.8-1.9.1) hosted at the Seven Bridge’s cloud platform (https://www.sevenbridges.com/). We utilized a custom mouse reference genome based on the Gencode version 25 (mm10) combined with our 500 sgRNA sequences. Since the BD pipeline uses STAR aligner in the backend, the custom STAR reference genome was generated by the genomeGenerate command in STAR (version 2.5.2b). The Refined Putative Cell Calling option was disabled while running the pipeline. Unique Molecular Identifier (UMI) count errors that are single-base substitution errors were identified and adjusted to the parent UMI barcode in the pipeline using RSEC (Recursive Substitution Error Correction) method, developed by BD Biosciences. The output consisted of thus corrected UMI counts, and was used for further downstream analysis.
Assembly: mm10
Supplementary files format and content: *_Expression_Data.st.gz compressed files contain a tab-separated (tsv) expression sparse matrix, in which each row records counts for cell-gene combinations that have non-zero RSEC molecule counts. Columns are the following: 1) Cell_Index: unique cell index number. 2) Gene: gene symbols. 3) RSEC_Reads: Number of reads after the RSEC molecular identifier adjustment algorithm. 4) Raw_Molecules: Number of UMIs before molecular identifier adjustment algorithms. 5) RSEC_Adjusted_Molecules: Number of UMIs after RSEC molecular identifier adjustment algorithm. *.h5 files are HDF5 files containing RSEC molecule counts with only the final subset of cells used in our publication.
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| Submission date |
Jun 20, 2023 |
| Last update date |
Jul 17, 2024 |
| Contact name |
Simona Baghai Sain |
| Organization name |
ETH Zurich
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| Department |
Department of Biosystems Science and Engineering
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| Street address |
Schanzenstrasse 44
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| City |
Basel |
| ZIP/Postal code |
4056 |
| Country |
Switzerland |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE235325 |
In vivo single-cell CRISPR uncovers distinct TNF-α programs in tumor evolution |
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| Relations |
| BioSample |
SAMN35814772 |
| SRA |
SRX20735158 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7500332_BD_WTA10_Expression_Data.st.gz |
321.3 Mb |
(ftp)(http) |
ST |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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