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| Status |
Public on Jul 10, 2020 |
| Title |
10x 10 cell lines Baselines |
| Sample type |
SRA |
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| Source name |
melanoma cell lines
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| Organism |
Homo sapiens |
| Characteristics |
tissue: melanoma cell lines
cell line: A375, MM001, MM011, MM029, MM031, MM047, MM099, MM074, MM087, MM057
treatment: no treatment
sequencing method: single cell RNA-Seq 10x
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| Treatment protocol |
The MM lines were cultured in Ham's F10 nutrient mix (ThermoFisher Scientific) supplemented with 10% fetal bovine serum (Invitrogen) and 100 µg ml-1 penicillin/streptomycin (ThermoFisher Scientific). The A375 cell line was maintained in Dulbecco’s Modified Eagle’s Medium with high glucose and glutamax (ThermoFisher Scientific), supplemented with 10% fetal bovine serum (Invitrogen) and penicillin-streptomycin (ThermoFisher Scientific). Cell cultures were kept at 37°C, with 5% CO2 and were regularly tested for mycoplasma contamination, and were found negative. Knockdown of SOX10 was performed using a SMARTpool of four siRNAs (SMARTpool: ON-TARGETplus SOX10 siRNA, number L017192-00, Dharmacon) at a final concentration of 20nM in Opti-MEM medium (Thermo Fisher Scientific) after which cells were sampled at the indicated time points. To control for transfection artefacts, we used a pool of non-targeting siRNAs (SMARTpool: ON-TARGETplus Non-targeting Pool, number D001810-10-05, Dharmacon). Inhibition of CDK7-dependent transcription was achieved by treatment with THZ2 (HY-12280, MedChemExpress). For each treated melanoma culture, IC50 concentrations were calculated for 48 hours of treatment. As a control, DMSO treatment was used. For bulk RNA-seq, RNA was extracted from attached cells using the innuPREP RNA mini kit (Analytik Jena), according to the manufacturer’s instructions. For scRNA-seq and Omni-ATAC-seq, cells were detached by trypsinization, counted and processed according to downstream protocols.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Not Provided
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
CellRanger v1.3.1; Demultiplexed with demuxlet
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| Data processing |
The 10x samples were processed (alignment, barcode assignment and UMI counting) with the CellRanger count pipeline; pools with samples from different cell lines were demultiplexed using demuxlet (Kang et al. 2018); cells expressing less than 1000 genes and cells with more than 20% mitochondrial reads were removed
The Drop-seq samples were cleaned with fastq-mcf from the ea-utils package (v1.04.807; Aronesty 2011) and processed (alignment, barcode assignment and UMI counting) with the Drop-seq tools pipeline (v1.12) developed by James Nemesh from the McCarroll lab (Macosko et al. 2015), using Picard tools (v1.140; Broad Institute) and the STAR aligner (v2.5.1b; Dobin 2013); cells expressing less than 1000 genes, more than 50000 and/or more than 10% mitochondrial reads were removed.
bulk RNA-Seq reads were cleaned with fastq-mcf from the ea-utils package (v1.04.807; Aronesty 2011), mapped using STAR (v2.5.1b; Dobin 2013) and counted with HTSeq (v0.6.1p1; Anders 2014); normalized count matrices were made with DESeq2 (v1.18.1; Love et al. 2014)
Omni-ATAC-Seq raw reads were mapped using Bowtie (v2.2.6; Langmead and Salzberg 2012); Duplicate reads were removed with Picard (v1.134; Broad Institute 2019) and low quality reads were removed with SAMtools (Q30) (v1.8; Li et al. 2009). bigWig files were created with deepTools (Ramírez et al. 2016).
Genome_build: hg19
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| Submission date |
Jul 17, 2019 |
| Last update date |
Jul 11, 2020 |
| Contact name |
k spanier |
| Organization name |
KU Leuven
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| Street address |
Herestraat 49
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| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE134432 |
Single-cell analysis of gene expression variation and phenotype switching in melanoma |
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| Relations |
| SRA |
SRX6457510 |
| BioSample |
SAMN12306971 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3946506_10x_A375_BL.tsv.gz |
2.3 Mb |
(ftp)(http) |
TSV |
| GSM3946506_10x_MM001_BL.tsv.gz |
1.7 Mb |
(ftp)(http) |
TSV |
| GSM3946506_10x_MM011_BL.tsv.gz |
1.3 Mb |
(ftp)(http) |
TSV |
| GSM3946506_10x_MM029_BL.tsv.gz |
2.2 Mb |
(ftp)(http) |
TSV |
| GSM3946506_10x_MM031_BL.tsv.gz |
1.9 Mb |
(ftp)(http) |
TSV |
| GSM3946506_10x_MM047_BL.tsv.gz |
1.4 Mb |
(ftp)(http) |
TSV |
| GSM3946506_10x_MM057_BL.tsv.gz |
1.9 Mb |
(ftp)(http) |
TSV |
| GSM3946506_10x_MM074_BL.tsv.gz |
1.4 Mb |
(ftp)(http) |
TSV |
| GSM3946506_10x_MM087_BL.tsv.gz |
2.0 Mb |
(ftp)(http) |
TSV |
| GSM3946506_10x_MM099_BL.tsv.gz |
2.0 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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