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Sample GSM4408002

Query DataSets for GSM4408002

Status

Public on Jul 10, 2020

Title

bulk MM087 TGFb/TNFa 72h

Sample type

SRA

Source name

MM_treatment with TGFbeta and TNFalpha 72h

Organism

Homo sapiens

Characteristics

cell line: MM087
cell type: melanoma cell line
treatment: treatment with TGFbeta and TNFalpha 72h
sequencing method: bulk RNA-Seq

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, regular tests for mycoplasma contamination were negative. Knockdowns of SOX10, EGR3 and NFATC2 were performed using a SMARTpool (Dharmacon) of four siRNAs, respectively (ON-TARGETplus SOX10 siRNA (L017192-00); ON-TARGETplus EGR3 siRNA (L006528-00); ON-TARGETplus NFATC2 siRNA (L003606-00)). SMARTpool was applied at final concentrations of 20nM (SOX10) and 40 nM (EGR3 and NFATC2) 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. Treatment with TGFbeta (10ng/ml; 100-21C, peprotech) and TNFalpha (1000 U/ml; 300-01A, peprotech) was performed in serum-free medium. As a control, also serum-free medium 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.

Extracted molecule

polyA RNA

Extraction protocol

not provided; requested

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

MM087_TGF_TNF_S11
processed data file:
bulkRNASeq_TGFb_TNFa_72h_raw_counts.tsv
bulkRNASeq_TGFb_TNFa_72h_normalized_counts.tsv

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
Supplementary_files_format_and_content: raw and normalized count matrices

Submission date

Mar 12, 2020

Last update date

Jul 10, 2020

Contact name

k spanier

Organization name

KU Leuven

Street address

Herestraat 49

City

Leuven