|
| Status |
Public on Apr 28, 2022 |
| Title |
I-miR-200c-3p_sorafenib_1 |
| Sample type |
SRA |
| |
|
| Source name |
HepG2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Hepatocellular carcinoma cells
cell line: HepG2
cell type: Hepatocellular carcinoma (HCC)
transfection: Inhibitor of miR-200c-3p 6h
treatment: Sorafenib 10 uM 6h
|
| Treatment protocol |
Sorafenib was added at the concentration of 10 µM at 24 h hours after transfection, and lysates were obtained after 6 and 24 hours after treatment. Control cells were treated in parallel with dimethyl sulfoxide (DMSO), vehicle in wich Sorafenib was dissolved.
|
| Growth protocol |
Cells were cultured in minimal essential medium (MEM) with Earle's balanced salts with L-glutamine suplemented with 10% fetal bovine serum, 1% sodium pyruvate, 1% non-essential amino acids and penicillin-streptomycin solution; cells were grown in culture flasks at 37 ºC in a humidified incubator with 5% CO2. Cells were plated at 50.000 cells/cm2 and transfected with Lipofectamine RNAimax after 24h with miR-200c-3p inhibitor, miR-222-5p mimic, miR-512-3p mimic and transfection controls. Media was changed 6 hours later
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using a miRNeasy mini kit with DNase treatment according to the manufacturer’s instructions(QIAGEN, Germany).
Libraries were prepared using total RNA lysates. Total RNA concentration and quality of the RNA were assessed with Qubit (Qubit™ DNA HS assay) and TapeStation 4150 with the RNA ScreenTape Assay (Agilent Technologies Genomics, USA), respectively. RNA Integrity Number (RIN) values were > 9 in all the RNA samples. Libraries were constructed from 350 ng of RNA with the QuantSeq 3' mRNA-Seq Library Prep Kit FWD for Illumina with unique dual indexes (set B1), folowing manufacturer instructions (Catalog number 114, Lexogen GmbH, Viena, Austria). Libraries were amplified (cycles 15-19), purified and subjected to quality control with High Sensitivity DNA Qubit® Assay and High Sensitivity D100 ScreenTape Assay (average size 318 bp). Samples were then pooled together at 2 nM, diluted at 750 nM (PhiX 2%) and sequenced in P2 flow cells in a NextSeq 2000 instrument from Illumina (65 cycles)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
I-miR-200c-3p sorafenibenib rep1
|
| Data processing |
Basecall and demultiplexing was carried out with DRAGEN FASTQ Generation online software (v. 3.8.4)
Secondary analysis, concerning alignment, mapping and differential expression analysis, was carried out using Bluebee® Quantseq pipeline
Reads were mapped to the human genome version GRCh38 using STAR Aligner with modified ENCODE settings
HTSeq-count was used for gene read counting
Differential expression analysis was performed using the DESeq2 method
Assembly: GRCh38
Supplementary files format and content: 2 Matrix tables with raw and normalized gene counts for every gene and every sample
|
| |
|
| Submission date |
Apr 27, 2022 |
| Last update date |
Apr 28, 2022 |
| Contact name |
Patricia de la Cruz-Ojeda |
| Organization name |
Institute of Biomedicine of Seville
|
| Lab |
209
|
| Street address |
Avda. Manuel Siurot s/n
|
| City |
Sevilla |
| ZIP/Postal code |
41011 |
| Country |
Spain |
| |
|
| Platform ID |
GPL30173 |
| Series (2) |
| GSE201695 |
Transcriptomic analysis of hepatocellular carcinoma cell lines HepG2 treated with Sorafenib and transfected with miR-200c-3p inhibitor, and miR-222-5p and miR-512-3p mimics |
| GSE201697 |
miR-200c-3p, miR-222-5p and miR-512-3p are outcome circulating biomarkers in patients with hepatocarcinoma under Sorafenib treatment |
|
| Relations |
| BioSample |
SAMN27916141 |
| SRA |
SRX15015601 |
