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Sample GSM3149745 Query DataSets for GSM3149745
Status Public on Aug 13, 2018
Title Cochlea SGNs Mouse 3
Sample type SRA
Source name WT mouse spiral ganglion cells
Organism Mus musculus
Characteristics strain: CBA/CaJ
age: 9 weeks
genotype/variation: wild-type
Sex: male
tissue: temporal bones
Extracted molecule total RNA
Extraction protocol Temporal bones from 9 wko, male CBA/CaJ mice (n = 8 capsules from 4 mice) were isolated in ice-cold Leibovitz’s L-15 medium, and the overlying bone and lateral wall was extracted from apex to base, leaving just the modiolus. The remaining structures consisted of the spiral ganglion, the spiral limbus, inner sulcus, and portions of the organ of Corti and outer sulcus. Microdissected tissue from each mouse was pooled in a solution of Leibovitz containing 200 units/mL collagenase IV (Sigma) and 10 kunitz/ml DNase I (Stem Cell Technologies) and incubated for 30 min at 37 °C to digest extracellular matrix. The collagenase IV solution was then replaced with EBSS containing 100 units of papain (Worthington Biochemical), 1.6 mM L-cysteine, 0.8 mM EDTA, 15 mM HEPES, and 10 kunitz/ml DNase I and incubated for an additional 30 min at 37 °C, triturating with a 1000 uL pipette every 10 min to generate a single cell suspension. An equal volume of Leibovitz containing 20% ovomucoid protease inhibitor (Worthington Biochemical) was added, and the dissociated cells were passed through a 20 mm filter (pluriSelect) to remove large debris. The cells were pelleted at 300g for 5 min, washed with PBS, and then pelleted and resuspended in PBS containing 0.1% BSA. Finally, a 10 uL sample of the cell suspension was counted on a Luna Fl automated counter using a Live/Dead assay (Thermo Fisher Scientific). Total time from euthanasia to single-cell capture was 2.5 h.
The cell suspension was diluted to a concentration of ~10,000 cells per uL and immediately captured, lysed, and primed for reverse transcription (RT) using the high throughput, droplet microfluidics Gemcode platform from 10X Genomics with v2 chemistry (Zheng et al., 2017). Each droplet on the Gemcode co-encapsulates a cell and a gel bead that is hybridized with oligo(dT) primers encoding a unique cell barcode and unique molecular identifiers (UMIs) in lysis buffer. The capture process takes 5 min, after which the transcriptomes captured on gel beads are pooled and immediately reverse transcribed to cDNA. Since all cDNA is pooled and PCR amplified, cell barcodes and UMIs facilitate demultiplexing of the originating cell and mRNA transcript after sequencing. RT, PCR amplification of cDNA, and preparation of a library from 3’ ends were conducted according to the manufacturer’s published protocol. The library was sequenced on an Illumina NovaSeq 6000 at the Broad Institute using 2 x 100 bp paired-end reads.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Description 3' polyA RNASeq reads
Data processing 3' polyA RNA-Seq (10X Genomics, chemistry v2)
CellRanger 2.0.1 (reference genome: mm10)
The gene count matrices were loaded into Seurat (v1.4) using the CreateSeuratObject function. Parameters used were min.cells=10, min.genes=100 and normalization.method="LogNormalize". Cells containing >5% red blood cell transcripts were removed.
Dimensional reduction (PCA) and clustering were performed using Seurat.
Clusters of cells expressing known spiral ganglion neuron markers (such as Nefh) were selected. Cells identified as type I spiral ganglion neurons were further subclustered to identify type I neuronal subtypes.
Genome_build: mm10
Supplementary_files_format_and_content: SGNs_rawCounts.txt : UMI counts (as calculated using CellRanger pipeline) showing the expression levels for each gene (rows) in each single cell (columns). The column header provides the cell barcode and the sample/mouse of origin separated by '_'.
Supplementary_files_format_and_content: SGNs_normCounts.txt : normalized counts (after Seurat processing) showing the expression levels for each gene (rows) in each single cell (columns). The column header provides the cell barcode and the sample/mouse of origin separated by '_'.
Supplementary_files_format_and_content: SGNs_metadata.txt : Table containing cell metadata. Columns are Cell (cell barcode and sample of origin, as appears in count matrices), NeuronType (indicates Type I or Type II spiral ganglion neuron identity) and NeuronSubtype (indicates Type I spiral ganglion subtype).
Submission date May 22, 2018
Last update date Aug 14, 2018
Contact name Gabriela Pregernig
Organization name Decibel Therapeutics
Street address 1325 Boylston Street, Suite 500
City Boston
State/province Massachusetts
ZIP/Postal code 02215
Country USA
Platform ID GPL24247
Series (1)
GSE114759 Transcriptional profiling of cochlear neurons
BioSample SAMN09237861
SRA SRX4112603

Supplementary file Size Download File type/resource
GSM3149745_SGNs_rawCounts_mouse3.txt.gz 6.1 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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