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| Status |
Public on Oct 02, 2022 |
| Title |
LFHT1_ROW23_9 |
| Sample type |
SRA |
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| Source name |
vomeronasal sensory neuron
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| Organism |
Mus musculus |
| Characteristics |
strain: B6;129P2-Omptm3Mom/MomJ
genotype: OMP(GFP/wt)
gender: male
age: 9-weeks-old
tissue: vomeronasal epithelium
cell type: vomeronasal sensory neuron
c1 capture chamber state: multiple cells in capture chamber
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| Extracted molecule |
total RNA |
| Extraction protocol |
3' end single-cell RNA sequencing using Fluidigm's C1 HT IFC with 5bp UMIs and 800 capture chambers
OMP(GFP/wt) males aged of 66 days were used (n=7). Mice were anesthetized using isoflurane and euthanized by decapitation. VNOs were dissected and their cartilaginous envelope were discarded. The tissues were immediately placed in ice-cold oxygenated artificial cerebro-spinal fluid (ACSF) with an osmolarity of 300 mOsm and pH at 7.4. After microdissection, the tissues extracted from the 7 mice were pooled into two tubes (i.e. 3 and 4 mice, respectively). Tissue dissociations were performed using the Papain Dissociation System (cat #LK003150, lot #35S16330; Worthington® Biochemical Corporation, New Jersey, USA) following the manufacturer’s protocol, with slight modifications; the EBSS solutions and the medium solution with serum were adapted from Saxena et al. (DOI: 10.2144/0000113878). To ensure the exclusive isolation of live nucleated cells, cell suspensions were incubated with 2 μg/ml of Hoechst 33342 (a UV fluorescent adenine-thymine binding dye; #H1399, Life Technologies) at 37°C for 15 min. In order to exclude dead cells, 1 μM of DRAQ7 (a far-red fluorescent DNA intercalating dye; #DR71000, BioStatus) was added to the cell suspensions before fluorescence activated cell sorting (FACS). Approximately 6000 GFP+/Hoechst+/DRAQ7- cells were collected from each single-cell suspension pool, each in a final volume of 15 μl. After FACS sorting, 4 μl of C1 Suspension Reagent (Fluidigm) was added to the 10 μl of cell suspensions, yielding mixes of 300 cells/μl. Cells were captured using the C1 Single-Cell mRNA Seq high-throughput (HT) integrated fluidic circuit (IFC) designed for 10-17 μm cells (cat #100-5760, Fluidigm). Each 14 μl mix was loaded on one side of the chip which was processed on the C1 System (cat #100-7000, Fluidigm) following the manufacturer’s protocol. Cell lysis, barcoding, reverse transcription and PCR amplification were performed directly on the C1 chip using Fluidigm’s C1 Single-Cell mRNA Seq HT Reagent Kit v2 (cat #101-3473, Fluidigm) following the manufacturer’s protocol. For a detailed description of the protocol, please refer to the methods section of Fodoulian et al. (2020) bioRxiv (https://0-doi-org.brum.beds.ac.uk/10.1101/2020.10.14.339259).
The cDNA content of each column on the chip (formed of 40 cells) was pooled before harvesting. Twenty cDNA libraries were generated using the Nextera XT DNA Library Preparation Kit (cat #FC-131-1024, Illumina) and Nextera XT Index Kit (cat #FC-131-1002, Illumina) following Fluidigm’s protocol. Before the final purification step, all 20 cDNA libraries were pooled. The library pool molarity and quality were assessed with the Qubit 2.0 using the Qubit dsDNA HS Assay Kit (cat #Q32854; Thermo Fisher Scientific) and the TapeStation using the Agilent High Sensitivity DNA Chip (cat #5067-5584; Agilent Technologies). The pool was then loaded at 7 pM on 4 lanes for clustering on a rapid paired-end Illumina Flow Cell (cat #PE-402-4002; Illumina) and sequenced on an Illumina HiSeq 2500 sequencer using the HiSeq Rapid SBS Kit v2 (cat #FC-402-4021; Illumina) chemistry. Read 1 consisted of 11 bases (6 bases for the cell barcode, and 5 bases for the unique molecular identifier), while read 2 was formed of 80 bases.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Two rounds of demultiplexing were performed on the raw Illumina data. The first round consisted in demultiplexing the Illumina indices using bcl2fastq2 Conversion Software version 2.20.0 (Illumina), which generated fastq files for each pool of 40 cells. The second round consisted in demultiplexing Fluidigm’s C1 HT IFC indices using the C1 mRNA Seq HT Demultiplexing Script version 1.0.2 (a Perl script from Fluidigm), which generated a pair of fastq files per cell (i.e. reads 1 and 2).
Digital gene expression matrices (i.e. matrices where genes are in rows, cells are in columns, and the corresponding gene counts are matrix entries) were generated following the single-cell analysis pipeline of UMI-tools version 0.5.3. First, unique molecular identifier (UMI) sequences were retrieved from read 1 and appended to the read names using the extract function from UMI-tools. The reads in read 2 (i.e. biological reads) were then mapped to the Ensembl release 86 of the Mus musculus genome reference 38 (GRCm38) using STAR version 2.5.4b. An updated version of the Ensembl release 86 GTF file of the mouse genome GRCm38 (mm10) was used. This custom gene annotation file included the manual re-annotation of the 3’ UTRs of the transcripts of vomeronasal receptor genes (V1rs, V2rs and Fprs), as well as the transcript annotations of de novo identified V1r pseudogenes. Multi-mapping of reads was allowed and the maximum number of hits was set to 4 (--outFilterMultimapNmax 4). For a detailed description, please refer to the methods section of the manuscript. Reads were then assigned to genes using featureCounts version 1.6.1 of the Subread package, and BAM files were sorted and indexed using SAMtools version 1.5. Deduplicated UMIs were then counted using the count function from UMI-tools. For each gene, UMIs that differed by 1 hamming distance were collapsed using the directional adjacency method (--method=directional --edit-distance-threshold=1 --per-gene --gene-tag=XT).
Genome_build: GRCm38
Supplementary_files_format_and_content: The vno_scRNAseq-e20170403_umi_tools_5bpUMI_pseudogenes_multimapping_fractional_LFJT20200404.txt file is a space-separated text file corresponding to the digital gene expression matrix of the single-cell RNA sequencing dataset. Rows are genes, columns are cells, and the corresponding gene counts (UMIs) are matrix entries. Column IDs correspond to sample titles and row IDs correspond to Ensembl mouse gene names.
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| Submission date |
Jan 04, 2022 |
| Last update date |
Oct 02, 2022 |
| Contact name |
Ivan Rodriguez |
| Organization name |
University of Geneva
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| Department |
Genetics and Evolution
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| Street address |
30 quai Ernest Anrsermet
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| City |
Geneva |
| ZIP/Postal code |
1205 |
| Country |
Switzerland |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE190613 |
Transcriptional instability prevents dispersion of functional vomeronasal receptor genes |
| GSE193024 |
Transcriptional instability prevents dispersion of functional vomeronasal receptor genes [scRNA-seq] |
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| Relations |
| BioSample |
SAMN24613705 |
| SRA |
SRX13602149 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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