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Sample GSM5360161

Query DataSets for GSM5360161

Status

Public on Aug 16, 2023

Title

HeLa shTra2b 1st

Sample type

SRA

Source name

ovarian cells

Organism

Homo sapiens

Characteristics

cell line: HeLa
transfection: shTra2b

Treatment protocol

To explore whether TRA2B could influence the invasive ability of ovarian cancer cells using the mothod of Transwell assay. The upper chamber was covered by100μl Matrigel (1mg/ml)(BD, USA). After Matrigel dried into gel, 200 μl cell suspension was added into transwell upper chamber and cultured for 24 hours at 37℃. Then, cells on the upper chamber were removed and cells from the lower surface were fixed with 7% ice ethanol, stained with 0.5% crystal violet for 30 mins. The cells were counted by ImagePro Plus.

Growth protocol

HeLa, HO8910 and A2780 cells were seeded in 24-well culture plates. When the cells reached 70% confluence, the cells were transfected with the vector using Lipofectamine 2000 according to the manufacturer's protocol. The cells were then incubated at 37℃ for 48 h, and viable cells were harvested and washed twice with phosphate-buffered saline (PBS). Viable cells were double-stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and 7-amino actinomycin D (7-AAD; Biotech Co., Ltd., Suzhou, Jiangsu, China). The percentage of cell apoptosis was defined as the sum of the right lower and upper quadrants. The induction of apoptosis was detected using the Annexin V/PI Apoptosis Detection Kit (Nan Jing Key Gen Biotech Co, Jiangsu, China) according to the manufacturer's instructions. Briefly, the cells were cultured at a density of 1.0×105 cells/mL, treated with TRA2B-shRNA/TRA2B-siRNA and cultured for 12 h at 37℃ with 5 % CO2. All samples were analyzed using a FACSort Flow Cytometer (Becton Dickinson, USA).

Extracted molecule

total RNA

Extraction protocol

The HeLa cells were ground into fine powder before RNA extraction. Total RNA was extracted with TRIzol (Ambion, Shanghai, China). The RNA was further purified with two phenol-chloroform treatments, then treated with RQ1 DNase (Promega, Madison, WI, USA) to remove DNA. The quality and quantity of the purified RNA were re-determined by measuring the absorbance at 260 nm/280 nm (A260/A280) using Smartspec Plus (BioRad, Hercules, California, USA). The integrity of RNA was further verified by 1.5% agarose gel electrophoresis.
For each sample, 1 μg of the total RNA was used for RNA-seq library preparation using the VAHTS Stranded mRNA-seq Library Prep Kit (Vazyme, Nanjing, Jiangsu, China). Polyadenylated mRNAs were purified and fragmented, then converted into double strand cDNA. After end repair and A tailing, the DNAs were ligated to VAHTS RNA Adapters (Vazyme). Purified ligation products corresponding to 200–500 bps were digested with heat-labile UDG, and the single strand cDNA was amplified, purified, quantified, and stored at -80℃before sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Description

expressed_gene_FPKM.txt; expressed_gene_reads.txt

Data processing

Raw reads containing more than 2-N bases were first discarded. Then, adaptors and low-quality bases were trimmed from raw sequencing reads using FASTX-Toolkit (version 0.0.13). The short reads less than 16nt were also dropped. The clean reads were then aligned to the GRch38 genome by TopHat2, allowing 4 mismatches. Uniquely mapped reads were used for gene read number counting and FPKM calculation (fragments per kilobase of transcript per million fragments mapped) .
The R Bioconductor packages edgeR and DESeq were utilized to screen out the DEGs. A false discovery rate <0.05 and fold change > 2 or < 0.5 were set as the cut-off criteria for identifying DEGs.
The alternative splicing events (ASEs) and regulated alternative splicing events (RASEs) between the samples were defined and quantified using the ABLas pipeline, as described previously. In brief, ABLas detection of 10 types of ASEs was based on the splice junction reads, including exon skipping (ES), alternative 5' splice site (A5SS), alternative 3'splice site (A3SS), intron retention (IR), mutually exclusive exons (MXEs), mutually exclusive 5'UTRs (5pMXEs), mutually exclusive 3'UTRs (3pMXEs), cassette exon, A3SS&ES, and A5SS&ES.
To assess RBP-regulated ASE, Student’s t-test was used to evaluate the significance of the ratio alteration of ASEs. Those events that were significant at the P value cut-off corresponding to a false discovery rate cut-off of 5% were considered RBP-regulated ASEs.
To sort out functional categories of DEGs, gene ontology (GO) terms and KEGG pathways were identified using the KOBAS 2.0 server. The hypergeometric test and Benjamin-Hochberg FDR controlling procedure were used to define the enrichment of each term.
Genome_build: GRch38
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for each Sample.

Submission date

Jun 05, 2021

Last update date

Aug 16, 2023

Contact name

Dong Chen

Organization name

ABLife, Inc.

Department

Center for Genome Analysis

Street address

388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone