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| Status |
Public on Sep 07, 2022 |
| Title |
Gallus gallus utricle, cell-269 |
| Sample type |
SRA |
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| Source name |
Utricle sensory epithelia
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| Organism |
Gallus gallus |
| Characteristics |
tissue: Utricle sensory epithelia
genotype: Wildtype
age: Posthatch day 7 (P7)
well plate: 4
plate position: G4
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| Extracted molecule |
total RNA |
| Extraction protocol |
Utricles were dissected in ice-cold Medium 199, otolithic membranes were mechanically removed, and tissue was incubated in thermolysin (0.5 mg/mL M199 medium) for 20 minutes at 37°C. Thermolysin activity was attenuated with 10% FBS in M199. The sensory epithelia were carefully peeled off from the underlying stromal cells using a 30- gauge 1⁄2-inch hypodermic needle attached to a 1 mL syringe. For each experiment, we pooled 4-8 sensory epithelia. The epithelia were dissociated using 1X Accutase for 20 minutes at 37°C, followed by mild mechanical trituration, and washed twice with PBS using a centrifugation step (300 x g, 5 min, room temperature (RT)). Viable single cells were isolated with a Becton Dickinson FACSAria Fusion flow cytometer (Ellwanger et al., 2018). Two independent batches of 270 cells were deposited into individual wells of 96-well plates, prefilled with 4 μl of a premade lysis solution with 1 U/μl of recombinant RNase inhibitor, 0.1% Triton X-100, 2.5 mM dNTP mix, 2.5 μM oligo d(T)30 VN (5’-AAGCAGTGGTATCAACGCAGAGTACT30VN-3’, IDT). Plates containing sorted cells were immediately sealed, frozen on dry ice, and stored at −80°C.
Single-cell RNA-seq was performed via Picelli and colleagues’ method (Picelli et al., 2014) using SMARTscribe for reverse transcription followed by 22 amplification cycles. Amplified cDNAs were purified by AMPure Beads cleanup using a Biomek FX automated platform and assessed with a fragment analyzer (Agilent) for quantitation and quality assurance. Barcoded libraries were synthesized using a scaled-down Nextera XT protocol (Mora-Castilla et al., 2016) in a total volume of 4 μl. A total of 384 libraries were pooled, and paired-end sequenced (2 x 150 bp) on a NextSeq 500/550 High Output flow cell.
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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 |
Smart-Seq2
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| Data processing |
Reads were aligned to the NCBI Gallus gallus v6.0 (GCF_000002315.6) reference genome and gene expression values were quantified with a customized STAR-RSEM pipeline for single-cell data.
FastQC and Scater were used for quality control.
Low-quality cells, ERCC spike-in transcripts, and low-level expressed genes were removed.
Assembly: NCBI Gallus gallus v6.0 (GCF_000002315.6) reference genome
Supplementary files format and content: Tab-separated values file reporting counts
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| Submission date |
Sep 07, 2022 |
| Last update date |
Sep 08, 2022 |
| Contact name |
Daniel C Ellwanger |
| Organization name |
Amgen Inc
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| Department |
Genome Analysis Unit
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| Street address |
1120 Veterans Blvd
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| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
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| Platform ID |
GPL19787 |
| Series (1) |
| GSE212831 |
Cell-type identity of the avian utricle |
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| Relations |
| BioSample |
SAMN30712555 |
| SRA |
SRX17467683 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6556161_cell-269_raw_counts.csv.gz |
55.6 Kb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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