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| Status |
Public on Aug 16, 2023 |
| Title |
HuBrain_Tumour_no437_S3 |
| Sample type |
SRA |
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| Source name |
GBM biopsy
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| Organism |
Homo sapiens |
| Characteristics |
tissue: GBM biopsy
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Single nuclei RNAseq: Protocol based on published nuclei isolation protocol (Södersten et al., 2018). Around 20 μg of -80 C-conserved tissue were thawed and dissociated in ice-cold lysis buffer (0.32M sucrose, 5 mM CaCl2, 3 mM MgAc, 0.1 mM Na2EDTA, 10 mM Tris-HCl pH 8.0, 1 mM DTT) using a 1 ml glass douncer (Wheaton). The homogenate was slowly and carefully layered in the centrifuge tubes on top of a sucrose layer (1.8 M sucrose, 3 mM MgAc, 10 mM Tris-HCl pH 8.0, 1 mM DTT) to create a gradient, and then centrifuged at 15500 rpm for 2 h 15 min. Following centrifugation, the supernatant was removed and the pellet softened for 10 minutes in 100 μl of nuclear storage buffer (15% sucrose, 10 mM Tris-HCl pH 7.2, 70 mM KCl, 2 mM MgCl2) prior resuspension in 300 μl of dilution buffer (10 mM Tris-HCl pH 7.2, 70 mM KCl, 2 mM MgCl2, Draq7 1:1000). The suspension was then filtered (70 μm cell strainer) and sorted via FACS (FACS Aria III, BD Biosciences) at 4° C with low flowrate, using a 100 μm nozzle (Pipette tips and Eppendorf tubes for transferring nuclei were pre-coated with 1% BSA).
Single nuclei RNAseq: 8500 nuclei were sorted for single-nuclei RNA-sequencing and then loaded onto the Chromium Next GEM Single Cell 3’ Kit (10x Genomics). Sequencing libraries samples were multiplexed and sequenced on a Novaseq machine using a 150-cycle kit using the recommended read length from 10x Genomics.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Single nuclei: Raw base calls were demultiplexed to obtained sample specific FastQ files using mkfastq in default parameters (10x Genomics), and reads were aligned to GRCh38 genome assembly using the Cell Ranger pipeline (10x Genomics, Cellranger count v3.1.0) with default parameters (--include-introns was used for nuclei mapping)
Assembly: GRCh38
Supplementary files format and content: Single cell/nuclei: Cellranger count output matrices
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| Submission date |
Jul 18, 2023 |
| Last update date |
Aug 16, 2023 |
| Contact name |
Mattias Belting |
| E-mail(s) |
mattias.belting@med.lu.se
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| Organization name |
Lund University
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| Department |
Clinical Sciences Lund
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| Lab |
Tumor Microenvironment
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| Street address |
Barngatan
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| City |
Lund |
| State/province |
Skåne |
| ZIP/Postal code |
SE-221 85 |
| Country |
Sweden |
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| Platform ID |
GPL24676 |
| Series (1) |
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| Relations |
| BioSample |
SAMN36534202 |
| SRA |
SRX21076298 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7635631_HuBrain_Tumour_no437_S3_barcodes.tsv.gz |
14.8 Kb |
(ftp)(http) |
TSV |
| GSM7635631_HuBrain_Tumour_no437_S3_features.tsv.gz |
297.6 Kb |
(ftp)(http) |
TSV |
| GSM7635631_HuBrain_Tumour_no437_S3_matrix.mtx.gz |
24.8 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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