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Sample GSM8267484

Query DataSets for GSM8267484

Status

Public on May 20, 2024

Title

PC3 cells,EphA2 gene knock out(infected with the CRISPR/Cas9 lentiviral vectors), Rep2

Sample type

SRA

Source name

prostate carcinoma

Organism

Homo sapiens

Characteristics

tissue: prostate carcinoma
cell line: PC3
cell type: Cancer cell line
genotype: EphA2 knockout
treatment: infected with the CRISPR/Cas9 lentiviral vectors

Treatment protocol

PC3 cells seeded in 6-well plates were infected with the CRISPR/Cas9 lentiviral vectors (EphA2-Cas9-sgRNA) and the control lentivirus carrying non-targeting sgRNA sequence. These cells were then incubated at 37℃ for 72 h.The efficiency of EphA2 gene knockout was verified by Sanger sequencing,then the EphA2 gene knockout efficiency was quantified using the Tracking of Indels by Decomposition (TIDE) online software (http://shinyapps.datacurators.nl/tide/).
DU145 cells were seeded in a 6-well plate. When the cells reached 80%-90% confluence, the EphA2 expression plasmid and control plasmid, respectively, were transfected into the cells using Lipofectamine TM 3000 transfection reagent (Invitrogen, USA) according to the manufacturer’s instructions. After incubation at 37℃ for 48 h, the efficiency of EphA2 overexpression was verified by quantitative real-time PCR (qPCR) and Western blotting.

Growth protocol

PC3 cells were maintained in Ham’s F-12K medium supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37℃.
DU145 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37℃.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted using Trizol reagent (thermofisher, 15596018) following the manufacturer's procedure. The total RNA quantity and purity were analysis of Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA, 5067-1511) , high-quality RNA samples with RIN number > 7.0 were used to construct sequencing library.
mRNA was purified from total RNA (5ug) using Dynabeads Oligo (dT) (Thermo Fisher, CA, USA) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature (Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94℃ 5-7min). Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA librarys were 300±50 bp. At last, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina NovaseqTM 6000 following the vendor's recommended protocol.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Reads obtained from the sequencing machines includes raw reads containing adapters or low quality bases which will affect the following assembly and analysis. Thus, to get high quality clean reads, reads were further filtered by Cutadapt (https://cutadapt.readthedocs.io/en/stable/,version:cutadapt-1.9). We aligned reads of all samples to the research species reference genome using HISAT2 (https://daehwankimlab.github.io/hisat2/,version:hisat2-2.2.1) package, which initially remove a portion of the reads based on quality information accompanying each read and then maps the reads to the reference genome.
The mapped reads of each sample were assembled using StringTie (http://ccb.jhu.edu/software/stringtie/,version:stringtie-2.1.6) with default parameters. Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare software(http://ccb.jhu.edu/software/stringtie/gffcompare.shtml,version:gffcompare-0.9.8). After the final transcriptome was generated, StringTie and ballgown (http://www.bioconductor.org/packages/release/bioc/html/ballgown.html) were used to estimate the expression levels of all transcripts and perform expression abundance for mRNAs by calculating FPKM (fragment per kilobase of transcript per million mapped reads) value.
Genes differential expression analysis was performed by DESeq2 software between two different groups (and by edgeR between two samples). The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 2 were considered differentially expressed genes. Differentially expressed genes were then subjected to enrichment analysis of GO functions and KEGG pathways.
Assembly: Homo_sapiens.GRCh38.101
Supplementary files format and content: tab-delimited text files include FPKM and count values for each Sample

Submission date

May 15, 2024

Last update date

May 20, 2024

Contact name

Chaogang Wei

E-mail(s)

20214133003@stu.suda.edu.cn

Organization name

The Second Affiliated Hospital of Soochow University

Street address

1055 Sanxiang Road, Suzhou 215004, Jiangsu, China