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Sample GSM8062769 Query DataSets for GSM8062769
Status Public on Feb 05, 2024
Title Cerebellum, NHP2
Sample type SRA
 
Source name cerebellum
Organism Macaca mulatta
Characteristics tissue: cerebellum
treatment: AAV9.UbC.trastuzumab
Treatment protocol Adult female Indian rhesus macaques (3–4 years old) were dosed via ICM injection with AAV9.UbC.trastuzumab
Extracted molecule nuclear RNA
Extraction protocol For nucleus isolation, buffers were typically prepared as described below, cooled in advance, and then supplemented immediately prior to use to make “complete” buffers at a final concentration of 1 mM dithiothreitol, 0.8 U/µl RNase inhibitor (Protector RNase Inhibitor; Roche, Indianapolis, IN, USA), and 1X protease inhibitor (complete Mini EDTA-free, Roche). For these assays ~25 mg of frozen tissue was minced with a scalpel and transferred to a pre-chilled 2-mL Dounce homogenizer with 1 mL of cold complete lysis buffer (0.32 M sucrose, 5 mM CaCl2, 3 mM magnesium acetate, 0.1 mM EDTA, 10 mM Tris-HCl pH 8.0, and 0.1% Triton X-100). The tissue was homogenized with 10 strokes each of pestle A then B and sequentially passed through pre-wetted 100-µM and 30-µM filters, with the filtered sample collected in a sterile tube. We washed the homogenizer and filters with an additional three volumes of complete lysis buffer, collected with the filtered sample. Two volumes of sample were then layered on top of one volume of cold complete isolation buffer (1.8 M sucrose, 3 mM magnesium acetate, and 10 mM Tris-HCl pH 8.0) and spun at 21 000g for 45 min at 4°C. We then carefully removed the supernatant and added 100 µL complete resuspension buffer (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, and 20 mM Tris-HCl pH 7.2) to the tube without mixing. We incubated the samples on ice for 10–15 min, fully resuspended the nuclei by gently pipetting up and down 20–30 times, and then counted the nuclei with an automated cell counter (Countess 3, Thermo Fisher). For submission of single-nucleus transcriptomic samples, we adjusted the concentration to ~1×106 nuclei/mL (range of 0.7–1.2×106 nuclei/mL) as per the manufacturer’s sample-loading guidelines.
Library preparation was performed according to the manufacturer's instructions (Single Cell 3' v3.1 protocol, 10x Genomics). Briefly, a single nuclei suspension was loaded alongside a master mix and gel beads. The gel beads and individual nuclei were partitioned into droplets to generate an emulsion. A unique molecular identifier and cell barcode were linked to poly-A RNA and reverse-transcribed into cDNA. The cDNA was recovered from the emulsion using silane Dynabeads, amplified to generate sufficient material for library preparation, and then size selected and purified post-amplification with SPRI-Select beads. A portion of the purified cDNA was then enzymatically fragmented, and partial Illumina adapters were ligated. A final PCR amplification further enriched the libraries and completed the Illumina adapters for sequencing, incorporating a unique sample index for demultiplexing
 
Library strategy RNA-Seq
Library source transcriptomic single cell
Library selection cDNA
Instrument model NextSeq 2000
 
Description 10X Genomics
Data processing Demultiplexing, barcode processing, and feature counting were all done using the 10X Genomics CellRanger software (v5.0.1)
Assembly: Mmul_10
Supplementary files format and content: cellrange output in MEX format, containing tab-delimited text files of cell barcode, feature (gene) annotation, and cell-feature read count
 
Submission date Feb 04, 2024
Last update date Feb 05, 2024
Contact name James M Wilson
Organization name University of Pennsylvania
Street address 125S 31st St
City Philadelphia
State/province Pennsylvania
ZIP/Postal code 19104
Country USA
 
Platform ID GPL32833
Series (1)
GSE255027 Adeno-associated virus-mediated trastuzumab delivery to the central nervous system for human epidermal growth factor receptor 2+ brain metastasis
Relations
BioSample SAMN39824384
SRA SRX23531062

Supplementary file Size Download File type/resource
GSM8062769_Sample3_barcodes.tsv.gz 72.8 Kb (ftp)(http) TSV
GSM8062769_Sample3_features.tsv.gz 289.9 Kb (ftp)(http) TSV
GSM8062769_Sample3_matrix.mtx.gz 151.3 Mb (ftp)(http) MTX
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