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| Status |
Public on Aug 15, 2023 |
| Title |
C3N-03186_CPT0206880004_snRNA_GBM |
| Sample type |
SRA |
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| Source name |
Brain
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Brain
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
15-25 mg of pulverized tissue was placed in a 5mL Eppendorf tube on ice. Using a wide-bore pipette tip (Rainin), a lysis buffer prepared from the Nuclei Isolation protocol (10X Genomics) and SuperRNase inhibitor (Invitrogen) was added to the tube. The tissue solution was gently pipetted until the lysis liquid turned a slightly cloudy color. (The number of pipetting iterations depended on the specific tissue.) The tissue homogenate was then filtered through a 40-micron strainer (pluriSelect) and washed with a BSA wash buffer (2% BSA + 1x PBS + RNase inhibitor). The filtrate was collected, centrifuged at 500 x g for 6 minutes at 4°C, and resuspended with a BSA wash buffer. 100 uL of cell lysis solution was set aside for unstained reference, while the rest was stained with DRAQ5 or 7AAD for RNA or ATAC sequencing, respectively. Namely, snRNA-seq nuclei were stained with 1 uL of DRAQ5 per 300 uL of the sample, and snATAC-seq nuclei were stained with 1 uL of 7AAD per 500 uL of the sample. Sorting gates were based on size, granularity, and dye staining signal. Nuclei and barcoded beads were isolated in oil droplets via the 10x Genomics Chromium instrument. Single nuclei suspensions were counted and adjusted to a range of 500 to 1800 nuclei/µL using a hemocytometer. Reverse transcription was subsequently performed to incorporate cell and transcript-specific barcodes.
All snRNA-seq samples were run using the Chromium Next GEM Single Cell 3’ Library and Gel Bead Kit v3.1 (10x Genomics). For snATAC-seq, Chromium Next GEM Single Cell ATAC Library and Gel Bead Kit v1.1 prep (10x Genomics) were used for all samples. Barcoded libraries were then pooled and sequenced on the Illumina NovaSeq 6000 system with specific flow cell types (snRNA-seq: S4; snATAC-seq: S1).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
10xV3.1 snRNA-seq
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| Data processing |
To process sequenced sc/snRNA-seq samples, Cell Ranger (v6.0.2) from 10X Genomics (with Count functionality) was used for aligning reads to the prebuilt GRCh38 genome reference version 2020-A (refdata-gex-GRCh38-2020-A) with the addition of pre-mRNA references. The Cellranger report from each sample was then carefully evaluated and we included samples with no critical errors or warnings. Examples of errors for which samples were excluded: “ERROR: Low Fraction Reads Confidently Mapped To Transcriptome”, or “ERROR: GEX Reads mapping to transcriptome is low.” Additionally, samples were excluded for less than 700 median genes per cell (except in certain cases where a high number of cells was detected).
To process sequenced snATAC-seq and snMutiome-seq data, we used the CellRanger-atac count (v2.0, 10X Genomics) and CellRanger-arc count (v2.0, 10X Genomics) pipelines, respectively. These pipelines filter and map snATAC-seq reads and identify transposase cut sites and the CellRanger-arc pipeline also performs filtering and alignment of snRNA-seq reads. The GRCh38 human reference was used for the read mapping. The CellRanger report from each sample was carefully evaluated and we excluded samples with few errors, except the “Number of cells is too high” error, while keeping samples with no errors or with just warnings. Examples of errors for which we removed samples are: “ATAC High-quality fragments in cells is low”, “ATAC TSS enrichment is low”, and “ATAC Fragments in peaks is low”.
Assembly: hg38
Supplementary files format and content: All files are standard Cellranger output files https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/output/overview or Cellranger-atac output files https://support.10xgenomics.com/single-cell-atac/software/pipelines/latest/output/overview
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| Submission date |
Aug 14, 2023 |
| Last update date |
Aug 15, 2023 |
| Contact name |
Alla Karpova |
| E-mail(s) |
a.karpova@wustl.edu
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| Organization name |
Washington University St Louis
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| Lab |
Li Ding Lab
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| Street address |
700 Rosedale avenue Campus Box 1000
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| City |
St Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE240822 |
Epigenetic regulation during cancer transitions across 11 tumour types. |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM7710020_C3N-03186_CPT0206880004_snRNA_GBM.tar.gz |
78.9 Mb |
(ftp)(http) |
TAR |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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