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Sample GSM6939175

Query DataSets for GSM6939175

Status

Public on Jan 27, 2024

Title

RCC4 VHL- JQ1 R1

Sample type

SRA

Source name

RCC4 VHL - JQ1

Organism

Homo sapiens

Characteristics

cell line: RCC4
genotype: loss of VHL
treatment: JQ1

Treatment protocol

RCC4 cells were treated with MYC inhibitor 5 μM JQ1. As control, DMSO was used. All compounds were from Sigma-Aldrich (St. Louis, MO, USA)

Growth protocol

The clear cell renal cell carcinoma cell line RCC4 with loss of VHL (RCC4 VHL) and the same cell line with VHL reintroduction (RCC4 VHL+) were maintained in high glucose DMEM with 10% FBS (HyClone, EU approved, heat Inactivated, South America) and 1% geneticin (Gibco, Life Technologies, Waltham, MA, USA). All cells were cultured in a humidified environment at +37°C and 5% CO2. RCC4 VHL+ and VHL- cells were transiently transduced with two different lentiviruses carrying one short hairpin RNA against MYC, shMYC1 (TRCN0000039640), shMYC2 (TRCN000003642), or both in combination shMYC1+2 (RCC4 VHL+ shMYC and RCC4 VHL- shMYC). As control, the cells were transduced with a lentivirus with the corresponding scrambled (SCR) control (RCC4 VHL+ shSCR and RCC4 VHL- shSCR). We used 4 μg/ml of polybrene and MOI=5 for both RCC4 VHL+ and RCC4 VHL- cells.

Extracted molecule

total RNA

Extraction protocol

RNA from cell lysates will be isolated using Tri-Reagent and RNA will be extracted and purified using DirectZol™ RNA MiniPrep kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s instructons.
DNase treatment was used to reduce the amount of genomic DNA. Ribosomal RNA (rRNA) was depleted from the samples using Ribosomal RNA depletion pools (riboPOOLs) developed by siTOOLs Biotech (Planegg, Germany), to enable sensitive detection of mRNA and non-coding RNA. RNA was reverse transcribed to cDNA fragmentation and size selection was performed to purify sequences that were the appropriate length for the sequencing machine. Once the RNA libraries were prepared, they were sequenced using a NextSeq 2000 instrument obtaining 30-40 million reads per sample.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 2000

Description

4_S4

Data processing

Reads were mapped to the Ensembl GRCh38 Homo Sapiens reference genome using STAR alignment software version 2.7.8 with Paired End setting
Raw abundance measurements were obtained using Subread package version 2.0.3 featureCounts feature with settings: -t "exon" -g "gene_id".
Differential expression analysis between JQ1 treatment versus DMSO control in both ccRCC4 VHL- and ccRCC4 VHL+ cell lines was done using DESeq2 version 1.30.1. Figures were created in RStudio using ggplot2 version 3.3.6.
The computations and data handling were enabled by resources in project SNIC 2021/22-252 provided by the National Academic Infrastructure for Supercomputing in Sweden (NAISS) at UPPMAX, funded by the Swedish Research Council through grant agreement no. 2022-06725.
Supplementary files format and content: tab-delimited text files include Raw Counts Table (pe counts for all 12 samples). And Differential expression Files (ccRCC4 VHL+ treated (JQ1) versus control (DMSO), as well as ccRCC4 VHL-).

Submission date

Jan 17, 2023

Last update date

Jan 27, 2024

Contact name

Irene Stevens

E-mail(s)

irene.stevens@scilifelab.se

Phone

0046728774966

Organization name

Karolinska Institutet