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Sample GSM5555729 Query DataSets for GSM5555729
Status Public on Feb 09, 2022
Title SiREV1#1-2
Sample type SRA
 
Source name A549_SiREV1#1
Organism Homo sapiens
Characteristics cell line: A549
cell type: Human lung adenocarcinoma cells
genotype: SiREV1#1
Treatment protocol A549 cells transfected with Scrambled or SiRNAs targeting REV1 for 48 hours.
Growth protocol All the cells were cultured in DMEM medium containing 10% FBS and 100 μg/mL penicillin and streptomycin in 5% CO2 at 37℃.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from the tissues using Trizol (Invitrogen, Carlsbad, CA, USA) according to manual instruction. About 60 mg of tissues were ground into powder by liquid nitrogen in a 2 mL tube, followed by being homogenized for 2 minutes and rested horizontally for 5 minutes. The mix was centrifuged for 5 minutes at 12,000×g at 4°C, then the supernatant was transferred into a new EP tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shacked vigorously for 15s, and then centrifuged at 12,000×g for 10 minutes at 4°C. After centrifugation, the upper aqueous phase where RNA remained was transferred into a new tube with equal volume of supernatant of isopropyl alcohol, then centrifuged at 13,600 rpm for 20 minutes at 4°C. After deserting the supernatant, the RNA pellet was washed twice with 1 mL 75% ethanol, then the mix was centrifuged at 13,600 rpm for 3 minutes at 4°C to collect residual ethanol, followed by the pellet air dry for 5-10 minutes in the biosafety cabinet. Finally, 25µL~100µL of DEPC-treated water was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA).
Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. Then First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. afterwards, A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular, DNBs were loaded into the patterned nanoarray and single end 50 bases reads were generated on BGIseq500 platform (BGI-Shenzhen, China).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model MGISEQ-2000RS
 
Data processing The RNA integrity was verified using Agilent 2100 Bioanalyzer (Agilent Technologies).
Paired-end libraries sequencing20 with Illumina MGISEQ-2000 were prepared with the mRNA-seq sample prep kit (Illumina) according to the manufacturer’s specifications.
Illumina data analysis pipeline was then used for sequence analysis. All samples were allocated to lanes and processed in blinded fashion to keep bias to a minimum
Analysis description: Calculate the expression read count using the Stringtie-1.3.3b software. Then, the gene and transcript FPKM were calculated. In this process we simply filtered the expression:1)Remove genes or transcripts with length less than 100 bp. ;2)If the FPKM of a gene or transcript is less than 0.0001, it is considered to be expressed as 0. Remove genes with a table of 0 in all samples.
Genome_build: hg38
Supplementary_files_format_and_content: abundance measurements
 
Submission date Sep 03, 2021
Last update date Feb 09, 2022
Contact name hua xiao jie
E-mail(s) m201775549@hust.edu.cn
Phone 15727070273
Organization name Huazhong University of Science and Technology
Department Tongji Medical College
Lab Cancer Center, Union Hospital,
Street address Xinhua Road
City Wuhan
State/province Hubei
ZIP/Postal code 430003
Country China
 
Platform ID GPL30209
Series (1)
GSE183332 REV1 promotes lung tumorigenesis by activating the Rad18/SERTAD2 axis
Relations
BioSample SAMN21212484
SRA SRX12007303

Supplementary file Size Download File type/resource
GSM5555729_si1d2.txt.gz 272.6 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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