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| Status |
Public on Oct 16, 2023 |
| Title |
Control_004_ribo |
| Sample type |
SRA |
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| Source name |
middle temporal gyrus
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| Organism |
Homo sapiens |
| Characteristics |
subject id: 004
tissue: middle temporal gyrus
age (yrs): 82
Sex: M
diagnosis: Control
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| Extracted molecule |
total RNA |
| Extraction protocol |
Frozen MTG was microdissected into ~5mm3 blocks. Frozen tissue was affixed to cryostat chucks with OTC and sectioned sectioned with a Microm HM525 Cryostat at -20ºC to 10 µm slices. For spatial transcriptomics samples, frozen tissue slides were adhered to the 10x Visium Spatial Gene Expression or Tissue Optimization slides following the manufacturer’s protocols. For bulk RNA-Seq, sections were adhered to Selectfrost microscope slides, and RNA was extracted using using the Direct-Zol RNA mini prep kit. All slides were frozen at -80ºC prior to library preparation.
For spatial transcriptomics, tissue optimization (i.e., permeabilization time) was tested according to the 10x Genomics protocols, with 12 min of permeabilization selected for spatial transcriptomics sequencing (data not shown). Two sections of each of the four subjects that were adhered to the 10X Visium Spatial Gene Expression were processed according to the 10x Genomics Visium protocols. Hematoxylin and eosin (H&E) imaging was performed at 20X magnification and auto-stitching using a Keyence BZ-X810 Microscope to generate high-resolution TIFF files. The cDNA was amplified using 12 cycles following Cq determination steps. Library size and quality was evaluated using the Agilent Tape Station-HS DNA Screen Tape and Tape Station; libraries were quantified with the KAPA qPCR library quantification kit. Spatial transcriptomics libraries were sequenced as paired-end 150-mer reads on an Illumina NovaSeq 6000 system using a NovaSeq S4 flowcell. Sequencing was performed by the USC Keck Genomics Platform (KGP). For bulk RNA-Seq, RNA was quantified and qualified using the Agilent Tape Station-HS RNA Screen Tape and Tape Station, and had an RNA Integrity Number (RIN) of 5.6. 240ng of RNA was used as input for each bulk RNA-Seq prep, which utilized the NEBNext Ultra II Directional RNA library prep kit. The polyA (mRNA) library preparation used the NEBNext Poly(A) mRNA Magnetic Isolation Module, and the ribo depletion library preparation used the NEBNext rRNA Depletion Kit V2 and SBP beads. Libraries were barcoded with the NEBNext Multiplex Oligos for Illumina. Library size and quality was evaluated using the Agilent Tape Station-HS DNA Screen Tape and Tape Station; libraries were quantified with the KAPA qPCR library quantification kit. Both RNA-Seq libraries were sequenced as paired-end 100-mer reads on an Illumina NovaSeq 6000 system using a NovaSeq S1 flowcell. Sequencing was performed by the USC Keck Genomics Platform (KGP).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
total RNA ribosomal depletion
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| Data processing |
For spatial transcriptomics, BAM files were generated using the 10x Genomics spaceranger pipeline (https://support.10xgenomics.com/spatial-gene-expression/software/overview/welcome). BAM files were converted to the three FASTQ files using the bamtofastq tool (https://support.10xgenomics.com/docs/bamtofastq).
For bulk RNA-Seq, paired-end FASTQ files (unprocessed) were used as input for the Splice-Break pipeline.
Assembly: NC_012920.1
Supplementary files format and content: top30matrix.txt: Tab-delimited text file which includes demographic information (diagnosis, sex, age), mitochondrial benchmark coverage, and deletion read % for each of the "Top 30" deletions and sum of the "Top 30" deletions. Deletion read %'s are after normalization to mitochondrial benchmark coverage.
Supplementary files format and content: top30matrix_ImputedLayers.txt: Tab-delimited text file which includes demographic information (diagnosis, sex, age), mitochondrial benchmark coverage, and deletion read % for each of the "Top 30" deletions and sum of the "Top 30" deletions for each imputed cortical layer from the spatial transcriptomics samples. Deletion read %'s are after normalization to mitochondrial benchmark coverage.
Supplementary files format and content: SpatialVISIUM_supp.tar.gz contains the following files for each spatial transcriptomics sample: (1) "SAMPLE_tissue_hires_image.png" = high resolution png images; (2) "SAMPLE_filtered_feature_bc_matrix.h5" = filtered feature gene by barcode count matrices in binary h5 format; (3) "SAMPLE_scalefactors_json.json" = JSON files with scaling factors for pixel positions in png images; (4) "SAMPLE_tissue_positions_list.txt" = text file with pixel coordinates for each barcode
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| Submission date |
Mar 05, 2023 |
| Last update date |
Oct 16, 2023 |
| Contact name |
Brooke Hjelm |
| E-mail(s) |
bhjelm@usc.edu
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| Organization name |
USC
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| Department |
Translational Genomics
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| Street address |
1450 Biggy Street
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90033 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE226663 |
Common Mitochondrial Deletions in RNA-Seq: Evaluation of Bulk, Single-Cell and Spatial Transcriptomic Datasets |
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| Relations |
| BioSample |
SAMN33588737 |
| SRA |
SRX19563977 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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