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| Status |
Public on Jan 16, 2021 |
| Title |
Probe5_A_375RR |
| Sample type |
SRA |
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| Source name |
Melanoma
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Melanoma cells
treatment: vemurafenib + cobimetinib
cell line: A375
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| Treatment protocol |
Cells were made resistant to vemurafenib or a combination of vemurafenib and cobimetinib or trametinib (MEK1/2 inhibitor) by keeping cells for 10-14 days under half IC50 concentrations of inhibitors, which had been determined in earlier experiments. Inhibitors were removed 24h before start of single-cell analyses in order to avoid direct toxic effects of substances on cell cultures. Chemical inhibitors were purchased from Biozol, Munich, Germany (vemurafenib, SEL-S1267; cobimetinib, SEL-S8041; trametinib, SEL-S2673).
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| Growth protocol |
The commercially available BRAF V600E mutant melanoma cell line A375 was used for the inhibitor experiments. Cells were kept under standard conditions in RPMI medium with 10% fetal calf serum and 1% penicillin/streptomycin.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell suspensions were generated by washing melanoma cells from different cultures with PBS followed by trypsinizing with TripLE Xpress (Invitrogen, Darmstadt, Germany) for 5 minutes. The reaction was stopped by adding media containing FCS. The number of dead cells was counted by Trypan blue staining (Sigma Aldrich, Munich) and did not exceed 5% of cells. Single melanoma cells from short-term cultures were captured on 10x Genomics Chromium Controller® according to the manufacturer’s specifications. 10,000 cells were used in these experiments. Overall, 10,000 cells were loaded into one channel of the Chromium system using the v2 single cell reagent kit (10x Genomics®). Following capture and lysis, cDNA was synthesized and amplified for 12 cycles.
The amplified cDNA was used to construct Illumina sequencing libraries according to the instruction of the Chromium Used Guide (10x Genomics). Briefly, blunt end repair was followed by adapter ligation. Finally, during PCR amplification sample indexes were added. The barcoded libraries were purified with SPRI beads and quantified using the Library Quantification Kit - Illumina/Universal (KAPA Biosystems) according to the instructions of the manufacturer. Correct size distribution of the library DNA was analyzed using the Fragment Analyzer (Agilent). A pool of up to 4 libraries was used for cluster generation.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Data processing |
Demultiplexing was done using bcl2fastq v2.20.0.422 (Illumina®).
For further analysis and read alignment CellRanger software was used with default settings suggested by 10x Genomics.
Genome_build: GRCh38
Supplementary_files_format_and_content: 4 matrix tables with raw gene counts for every gene and every sample (CellRanger output)
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| Submission date |
Jan 15, 2021 |
| Last update date |
Jan 17, 2021 |
| Contact name |
Maria Schmidt |
| E-mail(s) |
schmidt@izbi.uni-leipzig.de
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| Organization name |
IZBI
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| Street address |
Haertelstraße 16-18
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| City |
Leipzig |
| ZIP/Postal code |
04107 |
| Country |
Germany |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE164897 |
Single-Cell Trajectories of Melanoma Cell Resistance to Targeted Treatment |
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| Relations |
| BioSample |
SAMN17323653 |
| SRA |
SRX9856806 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5022597_raw_counts_probe5.txt.gz |
13.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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