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| Status |
Public on Mar 08, 2019 |
| Title |
SAM24348328: Untreated |
| Sample type |
SRA |
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| Source name |
melanoma tumor
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| Organism |
Mus musculus |
| Characteristics |
background: C57BL/6
tissue: Genetically engineered mouse model (GEMM) tumor
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| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were processed for single cell RNA sequencing (scRNAseq) using the Chromium Single Cell 3’ Library and Gel bead kit v2, following the manufacturer’s manual (10x Genomics, San Francisco, CA). Cell density and viability of the single-cell suspensions were determined by Vi-CELL XR cell counter (Beckman Coulter). All of the processed samples had very high percentage of viable cells. Cell density was used to impute the volume of single cell suspension needed in the reverse transcription (RT) master mix, aiming to achieve ~6,000 cells per sample. cDNAs and libraries were prepared following the manufacturer’s manual (10x Genomics, San Francisco, CA). Libraries were profiled by Bioanalyzer High Sensitivity DNA kit (Agilent Technologies, Santa Clara, CA) and quantified using Kapa Library Quantification Kit (Kapa Biosystems, Wilmington, MA). Each library was sequenced in one lane of HiSeq4000 (Illumina, San Diego, CA) following the manufacturer’s sequencing specification (10x Genomics, San Francisco, CA).
cDNAs and libraries were prepared according to manufacturer instructions (10× Genomics)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Illumina v1.8 software used for basecalling
The number of reads for all ~700k possible cell barcodes were tallied and data demultiplexed for cell barcodes represented by at least 10k reads. Transcript reads were aligned to the reference genome GRCm38 using GSNAP version ‘2013-10-10’ (parameters: ‘-M 2 -n 10 -B 2 -i 1 -N 1 -w 200000 -E 1); only uniquely mapping reads were considered. The number of transcripts per gene was inferred based on the number of unique molecular identifiers (UMIs) per gene (for reads overlapping exons in sense orientation) allowing for one mismatch between UMI sequences to account for errors in sequencing or PCR amplification. After excluding cells with <300 UMIs and removing non-expressed features, data were further processed using the Seurat R package version 2.2.0. Subsequent to normalization using the “LogNormalize” setting, data were scaled based on the total number of UMIs per cell. Then a principle component (PC) analysis was run on the most variably expressed genes and the first 30 PCs were used to perform t-distributed statistical neighbor embedding (tSNE) analysis and density clustering.
For each sample the R1 file provides the barcode representing the cell and the R2 file provides the insert sequence
Genome_build: mm10
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| Submission date |
Feb 18, 2019 |
| Last update date |
Mar 10, 2019 |
| Contact name |
Dorothee Nickles |
| E-mail(s) |
nicklesd@gene.com
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| Organization name |
Genentech
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| Street address |
1 DNA Way
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| City |
South San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94080 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE126714 |
Melanoma GEMM Tumors, untreated or vemurafenib treated (scRNASeq) |
| GSE205294 |
Melanoma GEMM Tumors, untreated or vemurafenib treated |
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| Relations |
| BioSample |
SAMN10962828 |
| SRA |
SRX5388928 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3611767_SAM24348328.txt.gz |
9.2 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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