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| Status |
Public on Jan 31, 2024 |
| Title |
PFA ependymoma, low cell density area, case 55, sample 3 |
| Sample type |
SRA |
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| Source name |
PFA ependymoma
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| Organism |
Homo sapiens |
| Characteristics |
tissue: PFA ependymoma
cell density_of_sample_region: low
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| Extracted molecule |
total RNA |
| Extraction protocol |
Posterior fossa type A (PF-EPN-A) tumor areas with both high and low cell density areas were selected on H&E-stained slides from FFPE tissues. Tissue sections (5 µm) of the corresponding areas were processed according to published protocols [Merritt et al. 2020, PMID: 32393914]. RNA-preserving antigen retrieval was performed, followed by RNA target exposure with proteinase K. In situ hybridization of all targets (n=18,677) on the tissue sections was performed overnight at 37°C, followed by stringent washes to remove off-target probes. Subsequently, antibodies against GFAP (Novus, NBP2-33184DL594, 5 µg/ml), Ki-67 (CST, 9027S, 6 µg/ml), and OLIG2 (Millipore, AB9610, 1:200) were used to visualize target areas as well as DAPI for nuclei counterstains. Three areas of low and high cell density each were selected with the custom polygon region of interest (ROI) tool for each case.
These selected areas were illuminated individually via UV light on a GeoMx DSP, resulting in photocleavage of oligonucleotides present within each ROI. Each aspirate contained photocleaved DNA oligos comprising an analyte identifier, a unique molecular identifier (UMI) barcode, and a primer binding site. Illumina adapter sequences and unique dual-sample indices were added when PCR was performed on the aspirates. Sequencing was performed using Illumina's NovaSeq 6000 with the S4 Reagent Kit v1.5 35 cycles flowcell, and paired end reads.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
PFA55_low_3
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| Data processing |
NanoString’s GeoMx NGS Pipeline v.2.3.4 was used to convert the fastsq files to Digital Count Conversion files. Quality control (QC) was performed using Nanostring GeoMx analysis suite. For #28 low, #41 high, #54 high and #59 high each, one out of the three samples had to be excluded after running QC due to a too low percentage of stitched and aligned reads and/or a too low negative probe count. Signals were scaled to nuclei count using the geometric mean method for each ROI and normalized for each probe against the 75th percentile of the cumulative signal of the respective ROI. Background correction was performed by geometric mean method against the average background calculated by signal to noise ratio using the negative probe counts.
Assembly: GRCh38.p13
Supplementary files format and content: ROI.dcc: Spatial Transcriptomics data as Digital Count Conversion files
Supplementary files format and content: RawCountMatrixafterQC_SpatialTranscriptomics_PFEPNA.xlsx: gene expression count data after QC in Excel table format, segment and target properties, dataset summary with processing parameters and annotations
Supplementary files format and content: ProcessedCountMatrix_SpatialTranscriptomics_PFEPNA.xlsx: gene expression count data after QC, scaling, normalization and background correction in Excel Table format, segment and target properties, dataset summary with processing parameters and annotations
Library strategy: Spatial Transcriptomics
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| Submission date |
Dec 01, 2023 |
| Last update date |
Jan 31, 2024 |
| Contact name |
Swenja Gödicke |
| Organization name |
Research Institute Children‘s Cancer Center Hamburg
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| Lab |
Schüller
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| Street address |
Martinistrasse 52
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| City |
Hamburg |
| ZIP/Postal code |
20251 |
| Country |
Germany |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE248811 |
Clinically relevant molecular hallmarks of PFA ependymomas display intratumoral heterogeneity and correlate with tumor morphology. |
| GSE249189 |
High and low cell density areas in posterior fossa ependymoma type A have a distinct transcriptome |
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| Relations |
| BioSample |
SAMN38579632 |
| SRA |
SRX22714704 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7927939_DSP-1012340078901-B-A11.dcc.gz |
67.2 Kb |
(ftp)(http) |
DCC |
SRA Run Selector |
| Raw data are available in SRA |
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