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| Status |
Public on Dec 23, 2020 |
| Title |
PJ052 |
| Sample type |
SRA |
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| Source name |
glioma surgical specimen
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| Organism |
Homo sapiens |
| Characteristics |
tissue: glioma surgical specimen
Sex: male
idh1 status: IDH1 mutant
diagnosis: WHO Grade IV Glioblastoma
primary/recurrent: primary
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
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 |
12-nt cell barcodes (CBs) and 9-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 beads associated with cells in our microwell system using the cumulative histogram of reads associated with each barcode
To avoid dead cells and library construction artifacts, we removed cell barcodes that failed to satisfy certain criteria. We removed all cells where >10% of molecules aligned to genes expressed from the mitochondrial genome or where the ratio of molecules aligning to whole gene bodies (including introns) to molecules aligning exclusively to exons was >1.5. These measures remove cells with compromised plasma membranes, which results in retention of mitochondrial or nuclear transcripts. We also removed cells where the number of reads per molecule (indicative of amplification efficiency) or the number of molecules per gene deviated by more than 2.5 standard deviations from the mean for a given sample.
Genome_build: GRCh38
Supplementary_files_format_and_content: Tab-delimited text files that contain the molecular counts of each individual cell. The first two columns contain the gene ID and gene symbol for each gene and all subsequent columns correspond to profiles of individual cells. There is a header contain the cell-identifying barcode sequence for each cell.
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| Submission date |
Dec 03, 2019 |
| Last update date |
Dec 24, 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) |
| GSE141383 |
Single-cell characterization of macrophages in glioblastoma reveals MARCO as a mesenchymal pro-tumor marker |
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| Relations |
| BioSample |
SAMN13473202 |
| SRA |
SRX7262715 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4202144_PJ052.counts.txt.gz |
3.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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