|
| Status |
Public on Mar 06, 2023 |
| Title |
RMC219_NEG_2 |
| Sample type |
SRA |
| |
|
| Source name |
Cell line derived from human renal medullary carcinoma (RMC219); see (Dong et al., 2017)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: RMC219
treatment: DMSO
tissue: Kidney
time after treatment: 0 hours
|
| Treatment protocol |
Genetically modified cells were cultured in adequate media supplemented with G418 (50uM) for selection. Reexpression experiments were done using 2uM doxycycline for the indicated amount of time.
|
| Growth protocol |
Fresh RMC219 were cultured for maximum 10 passages at a dilution of 1:1,5 in DMEM/F12 + 10% FCS + Glutamine 2mM + AANE + PS. Fresh RMC2C were cultured for maximum 10 passages at a dilution of 1:2 in MEM + 10% FCS + AANE + PS.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA-Seq libraries were generated from 600 ng of total RNA using TruSeq Stranded mRNA Library Prep Kit and TruSeq RNA Single Indexes kits A and B (Illumina, San Diego, CA), according to manufacturer's instructions. Briefly, following purification with poly-T oligo attached magnetic beads, the mRNA was fragmented using divalent cations at 94oC for 2 minutes. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved by replacing dTTP with dUTP during second strand cDNA synthesis using DNA Polymerase I and RNase H. Following addition of a single 'A' base and subsequent ligation of the adapter on double stranded cDNA fragments, the products were purified and enriched with PCR (30 sec at 98oC; [10 sec at 98oC, 30 sec at 60oC, 30 sec at 72oC] x 12 cycles; 5 min at 72oC) to create the cDNA library. Surplus PCR primers were further removed by purification using AMPure XP beads (Beckman-Coulter, Villepinte, France) and the final cDNA libraries were checked for quality and quantified using capillary electrophoresis.
RNA isolation was performed according to standard procedure (Macherey Nagel Nucleospin RNA Plus).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14.
Reads were preprocessed in order to remove adapter and low-quality sequences (Phred quality score below 20) using cutadapt version 1.10.
Reads were mapped to rRNA sequences using bowtie version 2.2.8, and reads mapping to rRNA sequences were removed for further analysis. Reads were mapped to spike sequences using bowtie2 version 2.2.8, and reads mapping to spike sequences were removed for further analysis.
Sequence reads were mapped to reference genome hg19 using Tophat v2.0.10 51 and the bowtie2 v2.1.0 aligner. Only uniquely aligned reads were retained for further analyses.
Gene expression quantification was performed from uniquely aligned reads using htseq-count version v0.6.1 with annotations from Ensembl version 75.
Data normalization and quantification of gene expression was performed using the DESeq2 Bioconductor package. Significantly deregulated genes were selected using a log2 fold change >1 and <1 and adjusted p-value cutoff of 0.05.
Genome_build: hg19
Supplementary_files_format_and_content: Excel file containing log2(fold-changes) and p-values computed by DESeq2 for SMARCB1 versus NEG comparisons at both timepoints in RMC219 and RMC-2C cells.
|
| |
|
| Submission date |
Jul 28, 2021 |
| Last update date |
Mar 06, 2023 |
| Contact name |
Guillaume Davidson |
| E-mail(s) |
davidsoy@igbmc.fr
|
| Organization name |
IGBMC
|
| Street address |
1 Rue Laurent Fries
|
| City |
Strasbourg |
| ZIP/Postal code |
67404 |
| Country |
France |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE180999 |
SMARCB1 regulates a TFCP2L1-MYC transcriptional switch promoting renal medullary carcinoma transformation and ferroptosis resistance. [RNAseq] |
| GSE181001 |
SMARCB1 regulates a TFCP2L1-MYC transcriptional switch promoting renal medullary carcinoma transformation and ferroptosis resistance. |
|
| Relations |
| BioSample |
SAMN20456163 |
| SRA |
SRX11582708 |
