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| Status |
Public on Jul 06, 2023 |
| Title |
HD376HD383, snRNAseq |
| Sample type |
SRA |
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| Source name |
Striatum
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| Organism |
Ovis aries |
| Characteristics |
cell type: Brain
tissue: Striatum
age: 5 year old
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| Extracted molecule |
total RNA |
| Extraction protocol |
Nuclei were extracted from frozen anterior dorsal ventral striatal (Figure 1) tissue utilising an adapted protocol from Krishnaswami et al., 2016 33. Briefly, approximately 50-100 mg of brain tissue was transferred to a dounce homogenizer containing 1mL homogenization buffer, HB (250mM sucrose, 25mM KCl, 5mM MgCl2, 10mM Tris buffer pH 8.0, 1µM DTT, 1X protease inhibitor (Sigma), 0.4 U/µL RNaseIn (ThermoFisher Scientific), 0.2 U/µL Superasein (ThermoFisher Scientific), 0.1 % Triton X-100). Tissue was homogenised using 5 strokes with the loose dounce pestle A, followed by 10-15 strokes of tight dounce pestle B. The homogenate was filtered through a 40 µm strainer into 5 mL Eppendorf tubes and centrifuged at 1000 rcf (4 oC) for 8 minutes. The supernatant was removed, and the pellet was resuspended in 250 µL of HB. A 50% - 29% iodixanol gradient (OptiPrep™ Density Gradient Medium, Sigma) was prepared to allow removal of the myelin layer. 250 µL of 50 % iodixanol was added to the nuclei-HB mixture and slowly layered on top of 500 µL of 29 % iodixanol in a new Eppendorf tube. The resultant gradient was centrifuged at 13,000 rcf (4oC) for 40 minutes. The supernatant and myelin was removed and the purified nuclei pellet was resuspended in 1 mL PBS, 1 % BSA, 0.2 U/µl RNAse inhibitor. 5 µL of resuspended nuclei was stained with 5 µL trypan blue and the quality and number of nuclei was assessed using the Countess II FL Automated Cell Counter (ThermoFisher Scientific).
Single nuclei RNA libraries were prepared using the Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 (10x Genomics) as per manufacturer’s instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
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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 CellRanger v7.0.0 pipeline with STAR v2.7.2a (https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger)
Sequencing ready libraries were sequenced on HiSeq Xten platform to generate on average 150 bp paired end reads. Alignment of reads was performed using the CellRanger v7.0.0 pipeline with STAR v2.7.2a to the sheep Oar_rambouillet_v1.0 reference genome and annotation (Ensembl release 107).
Assembly: Oar_Rambouilletv1.0
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Apr 16, 2023 |
| Last update date |
Jul 07, 2023 |
| Contact name |
Andrew Jiang |
| E-mail(s) |
ajia169@aucklanduni.ac.nz
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| Organization name |
The University of Auckland
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| Street address |
3 Symonds Street
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| City |
Auckland |
| ZIP/Postal code |
1010 |
| Country |
New Zealand |
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| Platform ID |
GPL23810 |
| Series (1) |
| GSE229839 |
Evidence for glutamate excitotoxicity that occurs before the onset of striatal cell loss and motor symptoms in an ovine Huntington’s Disease model. |
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| Relations |
| BioSample |
SAMN34209333 |
| SRA |
SRX19983165 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7178973_HD376HD383_barcodes.tsv.gz |
44.2 Kb |
(ftp)(http) |
TSV |
| GSM7178973_HD376HD383_features.tsv.gz |
201.1 Kb |
(ftp)(http) |
TSV |
| GSM7178973_HD376HD383_matrix.mtx.gz |
63.4 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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