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| Status |
Public on Dec 22, 2023 |
| Title |
Pretreatment NBL M228AAA_T, paired with M228AAA_T2, scRNAseq [M228AAA_T1_scR_CEL-Seq2_1] |
| Sample type |
SRA |
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| Source name |
NBL tumor left adrenal gland
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| Organism |
Homo sapiens |
| Characteristics |
tissue: NBL tumor left adrenal gland
tumor type: High-risk NBL
tumor stage: stage4
treatment: pre-treatment
Sex: female
genotype: MYCN non-amplified
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| Extracted molecule |
total RNA |
| Extraction protocol |
Tumor material was collected in the operating room by Truecut biopsy (samples at diagnosis) or surgical resection (debulking procedure after induction chemotherapy). The Dutch samples were gently dissociated into single‐cells and sorted by flow cytometry for single‐cell RNA sequencing using the CEL‐Seq2 platform. In brief, preparation of the tumor pieces for single‐cell RNA sequencing was started within 4 hours after the surgery. The material was minced into pieces <1mm3 and dissolved by enzymatic digestion with collagenase I, II and IV (2.5 mg/mL) at 37°C with agitation for max. 1 hour. The samples were filtered through a 70 μm cell strainer and washed in DMEM. Cells were further separated into a single cell suspension with the NeuroCult dissociation kit according to the manufacturer's protocol (StemcellTM Technologies, cat#05707), washed and stained with DAPI and DRAQ5. Next to this unbiased strategy, some samples were additionally stained with antibodies to enrich for T cell populations (NB124, NB125 with CD3‐PE (Biolegend, cat#300308); NB124 with TCRγδ‐BV421 (Biolegend, cat#331217) or with GD2‐FITC (BD Biosciences, cat#563439) to enrich for tumor cells (000GGU, 000GXF, NB106, NB107, NB098, NB125, NB125, NB130, NB152). Single live cells were sorted into 384‐well plates containing 10 μl of mineral oil, 50 nl of RT primers, deoxynucleotide triphosphates (dNTPs) and synthetic mRNA Spike‐Ins on a FACSJazz, FACSAria II or Sony SH800S machine and subsequently spun down, snap‐frozen on dry ice, and stored in ‐80⁰C to proceed with the total transcriptome amplification, library preparation and sequencing. Peripheral blood mononuclear cells (PBMC) from 5 healthy young adult donors (mean age 28) were isolated by Ficoll-Paque (VWR) density centrifugation and stained with fixable viability dye efluor506 (eBioscience, cat#65‐0866‐14), CD3‐PE (Biolegend, cat#300308) and CD56‐APC (Biolegend, cat#318310) antibodies. Subsequently, live CD3+ T cells and CD56+ NK cells were FACS sorted into 384‐well plates containing 10 μl of mineral oil, 50 nl of RT primers, deoxynucleotide triphosphates (dNTPs) and synthetic mRNA Spike‐Ins on a Sony SH800S machine. The plates were spun down, snap‐frozen on dry ice, and stored in ‐80⁰C to proceed with the total transcriptome amplification, library preparation and sequencing.
All samples were processed for total transcriptome amplification, library preparation and sequencing into Illumina sequencing libraries as previously described70. Paired‐end 2x75 bp sequencing read length was used to sequence the prepared libraries using the Illumina NextSeq sequencer. Sharq preprocessing and QC pipeline were applied to process the single‐cell RNA‐seq data as described71. Read mapping was done using STAR version 2.6.1 (RRID:SCR_004463) on the hg38 Patch 10 human genome. Function featureCounts (RRID:SCR_012919) of the subread package (version 1.5.2) was used to assign reads based on GENCODE version 26 (RRID:SCR_014966).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
CelSeq-2
barcodes.tsv
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| Data processing |
Failed reactions were identified by low levels of ERCC external RNA controls and excluded. Furthermore, a liveness threshold was calculated for each plate based on the wells with no cell added, in order to distinguish live cells from dead and/or apoptotic cells. This threshold was set to a minimum of 500 transcripts.
The percentage of transcripts mapping to the mitochondrial genome was calculated and cells with more mitochondrial‐encoded transcripts over nuclear ones were removed. Mitochondrial and ERCC transcripts were removed from the dataset, as well as cells with <1000 nuclear‐encoded transcripts or <500 genes. In addition, cells with >150.000 nuclear‐encoded transcripts were removed. Genes with low expression, that is either having less than 5 cells expressing the gene or less than two cells with less than two transcripts, were removed. A total of 105 cells, forming a distinct cluster of erythroid lineages, was identified based on high 515 levels of hemoglobin complex genes, and was removed. To improve cross‐sample comparisons, ambient mRNA contamination in individual cells was estimated and removed using DecontX. DecontX was run for all samples (batches) individually. Removal of cells with less than 1000 nuclear‐encoded transcripts was repeated on the decontaminated counts matrix outputted by DecontX.
All subsequent analyses were performed using R (version 4.0.2) and the package Seurat (version 3.2.2,RRID:SCR_007322) with default parameters unless stated otherwise. The SCTransform() function was used to normalize and scale the data, and to identify variable genes. To avoid clustering of cells based on specific cell processes, genes associated with sex (XIST, TSIX, and Y chromosome‐specific genes), cell cycle phase, dissociation stress (heat shock proteins; GO:0006986), and activity (ribosomal protein genes; GO:0022626), were removed as described before.
Assembly: hg38 Patch 10 human genome
Supplementary files format and content: Tab-separated values files and matrix files
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| Submission date |
Nov 15, 2022 |
| Last update date |
Dec 22, 2023 |
| Contact name |
Sander Roeland van Hooff |
| Organization name |
AUMC
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| Department |
Center for Experimental and Molecular Medicine (CEMM),
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| Lab |
Laboratory for Experimental Oncology and Radiobiology (LEXOR)
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| Street address |
Meibergdreef 9
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| City |
Amsterdam |
| ZIP/Postal code |
1105 AZ |
| Country |
Netherlands |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE218003 |
Gene expression profile at single cell level of neuroblastoma tumors and their tumor microenvironment |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM6732329_M228AAA_T1_scR_CEL-Seq2_1_counts.tsv.gz |
905.5 Kb |
(ftp)(http) |
TSV |
| GSM6732329_M228AAA_T1_scR_CEL-Seq2_1_features.tsv.gz |
223.4 Kb |
(ftp)(http) |
TSV |
| Processed data provided as supplementary file |
| Raw data not provided for this record |
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