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| Status |
Public on Dec 21, 2022 |
| Title |
humanDRG_sample16 |
| Sample type |
SRA |
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| Source name |
Dorsal Root Ganglion
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Dorsal Root Ganglion
donor #: Hom_5
age: 16 years
Sex: M
drg level: Lumbar 5
archival method: Frozen
nuclei isolation_method: Density gradient
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| Extracted molecule |
total RNA |
| Extraction protocol |
DRG samples from humans were first cut into ~1-2 mm pieces with a scalpel while placed on a dissection tray over dry ice and then placed in the lysis buffer (20 mM NaCl, 5 mM MgCL2, 0.1% TX-100, 10 mM Tris-HCl pH7.2) containing EDTA-Free protease inhibitor, RNAse inhibitor, and RNase-free DNase. Dounce homogenization was used to dissociate all DRG tissues. Before douncing, 1 mL HBSS containing 3% BSA Fraction VI and RNAse inhibitor in nuclei suspension buffer (NSB) was added to the 2 mL Dounce Tissue Grinder. The DRGs were homogenized with an A (“loose”) pestle using 5 to 10 strokes. The homogenate was then filtered through a 70 micron filter and spun down. The pellet was resuspended in the NSB. The frozen DRGs were not allowed to thaw before placing in the lysis buffer and douncing. From this homegenate, nuclei were isolated using two different methods: density-gradient centrifugation and FACS. For density-gradient centrifugation method, the homogenate was layered over an Optiprep density (40%, 20% and 10% for primates) gradient and centrifuged at 2500 g for 20 min. Nuclei at the 20/40 interface were aspirated and mixed with an equal volume of NSB. Nuclei were re-pelleted, washed, and counted using a hemocytometer prior to loading into a 10X Genomics Chromium controller. For FACS, nuclei from the density gradient were pelleted by centrifugation, labeled with propidium iodide and DAPI, and sorted using the Aria Fusion Flow Cytometer. Nuclei were selected based on double labeling with DAPI and PI and sorted into eppendorf tubes containing 0.5 mL nuclei suspension buffer (NSB). Nuclei were then counted, pelleted and resuspended prior to loading into a 10X Genomics Chromium controller.
Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (10X Genomics) were used for library preparation according to the manufacturer’s user guides. The Cell-RT mix was prepared to aim for 10,000 nuclei per sample and applied to the ChromiumTM Controller for GEM generation and barcoding. Then samples were subjected to post GEM-RT cleanup, cDNA amplification (11 cycles with v3.1), and library construction according to the user manual. Sample index PCR was done with 12 cycles. Libraries were then quantified by Qubit dsDNA HS Assay Kit and profiled by Bioanalyzer High Sensitivity DNA kit.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
10X genomics
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| Data processing |
The demultiplexing, barcoded processing, gene counting and aggregation were made using the Cell Ranger software v5.0.0 and v2.4.0 (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Assembly: hg19
Supplementary files format and content: h5 files
Supplementary files format and content: humanallcelltypes.rds: Human Seurat object with cell type annotations for all cell types.
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| Submission date |
Apr 26, 2022 |
| Last update date |
Feb 06, 2023 |
| Contact name |
Josh Kaminker |
| E-mail(s) |
kaminker@gene.com
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| Organization name |
Genentech Inc
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| Department |
OMNI Bx, gRED
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| Street address |
1 DNA Way
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| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE201586 |
Cross-species single-nuclei transcriptome atlas of dorsal root ganglion sensory neurons [humanDRG] |
| GSE201654 |
Cross-species single-nuclei transcriptome atlas of dorsal root ganglion sensory neurons |
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| Relations |
| BioSample |
SAMN27782900 |
| SRA |
SRX15003471 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6068108_SAM24403167_filtered_feature_bc_matrix.h5 |
20.5 Mb |
(ftp)(http) |
H5 |
| GSM6068108_SAM24403167_raw_feature_bc_matrix.h5 |
65.1 Mb |
(ftp)(http) |
H5 |
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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