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| Status |
Public on Jul 13, 2022 |
| Title |
SF11857_spatial_proteomics |
| Sample type |
SRA |
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| Source name |
Brain
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Brain
progression: Recurrent
diagnosis: GBM
age: 69
gender: Male
pair#: SF10592
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| Extracted molecule |
protein |
| Extraction protocol |
For the spatial transcriptomics assay, FFPE tissue blocks were reviewed for tumor purity and 10, 5 µm sections were cut by the UCSF Neurosurgery Tissue Core. Slides were baked at 37°C overnight and then deparaffinized, rehydrated, antigen-retrieved for 20 min at 100°C, and digested with proteinase-K 0.1 ug/ml for 15 min in a Leica BOND-RX. Samples were post-fixed in neutral-buffered formalin for 10 min and hybridized to the cancer-transcriptome atlas (>1,800 targets) UV-photocleavable barcode-conjugated RNA in situ hybridization probe set overnight. Samples were washed to remove off-target probes and counterstained with morphology markers for 2 hours. The morphology markers consisted of: 1:25 SYTO13 (Invitrogen), 1:100 anti-CD3 Alexa Fluor 647 (UMAB54, Origene)), 1:200 anti-CD68-Alexa Fluor 594 (KP1, Santa Cruz), and 1:400 anti-GFAP-Alexa Fluor 488 (GA5, Invitrogen). Immunofluorescence imaging, region of interest (ROI) selection, spatially-indexed barcode cleavage and collection were performed on a GeoMx Digital Spatial Profiling instrument (NanoString). Approximately 12 ROIs were collected per sample. Photoreleased GeoMx DSP oligonucleotide tags containing RNA IDs and a UMI were collected from each ROI. After PCR with dual-indexing Illumina i5 and i7 primers, the library was purified with AMPure XP beads (Beckman Coulter), quantitated with a Qubit (Themo Fisher Scientific) and quality was checked with a Bioanalyzer (Agilent). Paired-end sequencing was performed on NextSeq 550 and NextSeq 2000 instruments. For the spatial proteomics assay, FFPE tissue blocks were reviewed for tumor purity and 6, 5 µm sections were cut by the UCSF Neurosurgery Tissue Core. Slides were baked at 37°C overnight, deparaffinized, rehydrated, antigen-retrieved in a pressure cooker for 15 min at 100°C at high pressure. Samples were then incubated overnight with the GeoMx Immune Cell Profiling Protein Core antibodies (NanoString) containing UV-photocleavable barcode-conjugated antibodies against 17 targets and 6 control targets. At the same time, the samples were incubated with morphology antibodies consisting of: SYTO83 (100 nM final concentration), 1:200 anti-CD68-Alexa Fluor 594 (clone KP1), 1:200 anti-CD45-Alexa Fluor 647 (clone 2B11+PD7/26), and 1:400 anti-GFAP-Alexa Fluor 488 (clone GA5). Immunofluorescence imaging, region of interest (ROI) selection, spatially-indexed barcode cleavage and collection were performed on a GeoMx Digital Spatial Profiling instrument (NanoString) by GENEWIZ. Approximately 10 ROIs were collected per sample. The resulting photocleavable barcode tags were subsequently detected and counted using an nCounter Prep Station and Digital Analyzer (NanoString).
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| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Data processing |
The SP data were normalized by ROI surface area. The ST data were normalized against the 75th percentile of signal, i.e. Q3 normalization. MUlti-Subject SIngle Cell deconvolution (MuSiC) was used for deconvolution of the spatial transcriptomics data based on cell-type signatures determined from snRNA-seq data used as input. Heatmaps were generated via pheatmap package (https://cran.r-project.org/web/packages/pheatmap/index.html). To validate putative ligand-receptor interactions via ST, the expression of each ligand-receptor pair obtained from CellChat were used to calculate a Pearson's correlation coefficient across ROIs. These were adjusted for multiple-hypothesis testing via fdrtool. Representative IF images for specific pathways were obtained based on sorting for correlation and then for expression of ligand-receptor pairs in that pathway.
Assembly: hg38
Supplementary files format and content: Read count; image data
Library strategy: Spatial proteomics
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| Submission date |
Jul 13, 2022 |
| Last update date |
Jul 13, 2022 |
| Contact name |
Aaron Diaz |
| E-mail(s) |
aaron.diaz@ucsf.edu
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| Organization name |
University of California, San Francisco
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| Department |
Neurological Surgery
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| Lab |
Diaz Lab
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| Street address |
1450 3rd St
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE174554 |
A single cell atlas of human glioma under therapy |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM6337078_SF11857_Proteomics.zip |
80.4 Mb |
(ftp)(http) |
ZIP |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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