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| Status |
Public on Apr 18, 2020 |
| Title |
PW029-702 |
| Sample type |
SRA |
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| Source name |
acute slice culture of glioma resection
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| Organism |
Homo sapiens |
| Characteristics |
tissue: acute slice culture of glioma resection
age: 52
gender: F
location: splenial extension into left parietal
diagnosis: Glioblastoma, WHO Grade IV
treatment: 2.5 uM etoposide
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| Growth protocol |
Tumor specimens were collected immediately after surgical removal and kept in ice-cold artificial cerebrospinal fluid (ACSF) solution containing 210 mM sucrose, 10 mM glucose, 2.5 mM KCl, 1.25 mM NaH2PO4, 0.5 mM CaCl2, 7 mM MgCl2 and 26 mM NaHCO3 for transportation. Preparation of ex vivo tissue slices was modified from methods described previously4. Briefly, the collected tumor specimens were placed in a drop of ice-cold ACSF and sliced using a tissue chopper (McIlwain) at a thickness of 500 µm under sterile conditions. The slices were immediately transferred to the ice-cold ACSF solution in 6-well plates using a sterile plastic Pasteur pipette half filled with ice-cold ACSF solution followed by a 15 minutes recovery in ACSF to reach room temperature. Intact and well-shaped slices (approximately 5-10 mm diameter) were then placed on top of a porous membrane insert (0.4 µm, Millipore). Then the membrane inserts were placed into 6-well plates containing 1.5 mL maintenance medium consisting of F12/DMEM (Gibco) supplemented with N-2 Supplement (Gibco) and 1% penicillin/streptomycin (Sigma). To ensure proper diffusion into the slice, culture medium was placed under the membrane insert without bubbles. A drop of 10 µl of culture medium was added directly on top of each slice to prevent the slice surface from drying. The slices were first rested for 6 hrs with the maintenance medium in a humidified incubator at 37℃ and 5% CO2. Then, the medium was replaced with pre-warmed medium containing drugs with desired concentration (Table S1) or corresponding volume of vehicle (DMSO). Slices were then cultured with the treatment medium in a humidified incubator at 37℃ and 5% CO2 for 18 hrs before being collected for dissociation.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Collected tissue samples or tissue slices were dissociated using the Adult Brain Dissociation kit (Miltenyi Biotec) on gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec) according to the manufacturer’s instructions.
mRNA molecues were captured using bead-bound oligo dT(30).
Captured mRNA molecues were reverse transcriped to obtain cDNA which is then amplified by PCR reactions. The cDNA PCR reaction product was then purified with AMPure XP beads. The purified cDNA PCR product was used as the input for Nextera XT reactions to generate sequencing library.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Read 1 and read 2 fastq files were generated on basespace.
Raw data obtained from the Illumina NovaSeq 6000 was first corrected for index swapping to avoid cross-talk between sample index sequences using the algorithm described by Griffiths et al, Nature Communications, 2018 before assigning read addresses for each sample.
12-nt cell barcodes (CBs) and 8-nt unique molecular identifiers (UMIs) were extracted from read 1. Degenerate CBs containing either 'N's or more than four consecutive 'G's were discarded. Synthesis errors, which can result in truncated 11-nt cell barcodes, were corrected.
Read 2 were trimmed to remove poly(A) tails (> dA(7)) from the 3'-ends of each read. Any resulting fragment shorter than 24 nucleotides were discarded.
Trimmed reads were then aligned to GRCh38 (GENCODE v.24) using STAR v.2.5.0 with parameters "-- sjdbOverhang 65 --twopassMode Basic --outSAMtype BAM Unsorted". Only reads with unique, strand-specific alignments to exons were kept for further analysis.
All reads with the same CB, UMI, and gene mapping were collapsed to represent a single molecule. To correct for sequencing errors in UMIs, we further collapsed UMIs that were within Hamming distance 1 of another UMI with the same barcode and gene. To correct for sequencing errors in cell barcodes, we then collapsed CBs that were within Hamming distance one of another cell barcode, had at least 20 unique IMI-gene pairs, and had at least 75% overlap of their UMI-gene pairs. A digital gene expression matrix was then generated from these corrected barcode-UMI-gene triplets.
We estimated the number of cell barcodes corresponding to beads associated with cells in our microwell system using the emptyDrops algorithm from Lun et al, Genome Biology, 2019.
We removed CBs that satisfied any of the following criteria 1) fractional alignment to the mitochondrial genome greater than 1.96 standard deviations above the mean, 2) ratio of molecules aligning to whole gene bodies (including introns) to molecules aligning exclusively to exons greater than 1.96 standard deviations above the mean, 3) average number of reads per molecule or average number of molecules per gene >2.5 standard deviations above the mean for a given sample, 4) more than 40% of UMI bases are T or where the average number of T-bases per UMI is at least 4..
Genome_build: GRCh38
Supplementary_files_format_and_content: Tab-delimited text files that contain the gene expression level of each individual cell.
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| Submission date |
Apr 17, 2020 |
| Last update date |
Apr 20, 2020 |
| Contact name |
Peter A Sims |
| E-mail(s) |
pas2182@columbia.edu
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| Organization name |
Columbia University
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| Street address |
3960 Broadway, Lasker 203AC
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE148842 |
Deconvolution of Cell Type-Specific Drug Responses in Human Tumor Tissue with Single-Cell RNA-seq |
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| Relations |
| BioSample |
SAMN14612003 |
| SRA |
SRX8131122 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4483742_PW029-702.cts.txt.gz |
10.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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