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Status |
Public on Feb 09, 2022 |
Title |
SiREV1#1-2 |
Sample type |
SRA |
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Source name |
A549_SiREV1#1
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Organism |
Homo sapiens |
Characteristics |
cell line: A549 cell type: Human lung adenocarcinoma cells genotype: SiREV1#1
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Treatment protocol |
A549 cells transfected with Scrambled or SiRNAs targeting REV1 for 48 hours.
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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℃.
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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).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
MGISEQ-2000RS |
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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
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Submission date |
Sep 03, 2021 |
Last update date |
Feb 09, 2022 |
Contact name |
hua xiao jie |
E-mail(s) |
m201775549@hust.edu.cn
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Phone |
15727070273
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Organization name |
Huazhong University of Science and Technology
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Department |
Tongji Medical College
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Lab |
Cancer Center, Union Hospital,
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Street address |
Xinhua Road
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City |
Wuhan |
State/province |
Hubei |
ZIP/Postal code |
430003 |
Country |
China |
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Platform ID |
GPL30209 |
Series (1) |
GSE183332 |
REV1 promotes lung tumorigenesis by activating the Rad18/SERTAD2 axis |
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Relations |
BioSample |
SAMN21212484 |
SRA |
SRX12007303 |
Supplementary file |
Size |
Download |
File type/resource |
GSM5555729_si1d2.txt.gz |
272.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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