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Sample GSM8229351 Query DataSets for GSM8229351
Status Public on May 18, 2024
Title KTC1 cells,TGFBR3 _Over-expression-1,rep1
Sample type SRA
 
Source name Thyroid
Organism Homo sapiens
Characteristics tissue: Thyroid
cell line: KTC1
cell type: Papillary thyroid cancer cells
genotype: Overexpression TGFBR3
Extracted molecule total RNA
Extraction protocol Add 200ul chloroform for every 1mlTRIzol, swirl and mix for 30 seconds, and let stand at room temperature for 3 minutes; Then centrifuge (4℃, 12000g×15 min) and slowly draw the upper water phase to the new 1.5ml EP tube. Add 500ul isopropyl alcohol for every 1ml of TRIzol, mix upside down, and let stand at room temperature for 10 minutes; After centrifugation, the supernatant was discarded (4℃, 12000g×10 min) to obtain RNA precipitation: the EP tube was inverted on the toilet paper and placed in a ventilated place to dry the RNA precipitation. Every 1ml of TRIzol was added with 1ml 75% ethanol, and then blown and mixed well. After centrifugation, discard the supernatant (4℃, 7500g×5 minutes), reverse the EP tube on the toilet paper, and place it in the fume hood to dry. Add nuclease-free water and mix well. Then heat at 55-60℃ for 10 minutes. The concentration and purity of RNA were determined by ultraviolet spectrophotometer to determine the light absorption of total RNA at 260nm and 280nm wavelengths.
Total RNA was extracted from the samples by Trizol reagent (Invitrogen) separately. The RNA quality was checked by Agilent 2200 and kept at −80°C. The RNA with RIN (RNA integrity number) > 7.0 is acceptable for cDNA library construction. The cDNA libraries were constructed for each RNA sample using the TruSeq Stranded mRNA Library Prep Kit (Illumina, Inc.) according to the manufacturer’s instructions. Generally, the protocol consists of the following steps: Poly-A containing mRNA was purified from 1ug total RNA using oligo(dT) magnetic beads and fragmented into 200-600 bp using divalent cations at 85℃ for 6 min. The cleaved RNA fragments were used for first- and second-strand complementary DNA (cDNA) synthesis . dUTP mix was used for second-strand cDNA synthesis, which allows for the removal of the second strand. The cDNA fragments were end repaired, A-tailed and ligated with indexed adapters. The ligated cDNA products were purified and treated with uracil DNA glycosylase to remove the second-strand cDNA. Purified first-strand cDNA was enriched by PCR to create the cDNA libraries. The libraries were quality controlled with Agilent 2200 and sequenced by NovaSeq 6000 on a 150 bp paired-end run.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description TGFBR3_Over-expression-1
Data processing RNA mapping:In this RNA-SEQ data analysis process, we used Hisat2 as the RNA mapping algorithm to compare the filtered clean data to the sequenced species (Taxonomy ID: 9606) on the corresponding reference genome (GRCh38) to determine the location of sequencing sequences on the genome.
Expression statistics:According to the results of RNA Mapping and genome annotation file (detailed recording of the location information of each gene on the chromosome), HTSeq algorithm was used to calculate the expression of each gene.
We applied DESeq2 algorithm [Love M I , Huber W , Anders S . Moderated Estimation of Fold Change and Dispersion for RNA-Seq Data with DESeq2[J]. Genome Biology, 2014, 15(12):550.] to filter the differentially expressed genes, after the significant analysis, P-value and FDR analysis [Benjamini Y, et al. Controlling the false discovery rate in behavior genetics research. Behav Brain Res. 2001 Nov 1;125(1-2):279-84.] were subjected to the following criteria: i) Fold Change>2 or < 0.5; ii) ,P-value<0.05,FDR<0.05.
Gene ontology (GO) analysis was performed to facilitate elucidating the biological implications of the differentially expressed genes in the experiment. [Ashburner M, et al. Gene ontology: tool for the unification of biology. The Gene Ontology Consortium. Nat Genet. 2000 May;25(1):25-9.]. We downloaded the GO annotations from NCBI (http://www.ncbi.nlm.nih.gov/), UniProt (http://www.uniprot.org/) and the Gene Ontology (http://www.geneontology.org/). Fisher’s exact test was applied to identify the significant GO categories (P-value < 0.05).
Pathway analysis was used to find out the significant pathway of the differentially expressed genes according to KEGG database. We turn to the Fisher’s exact test to select the significant pathway, and the threshold of significance was defined by P-value< 0.05. [Draghici S, et al. A systems biology approach for pathway level analysis. Genome Res. 2007 Oct;17(10):1537-45. Epub 2007 Sep 4.]
Assembly: GRCh38.Ensembl104
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample
Supplementary files format and content: tab-delimited text file includes FPKM for each Sample
 
Submission date Apr 24, 2024
Last update date May 18, 2024
Contact name zhang han rong
E-mail(s) zhanghr68@mail2.sysu.edu.cn
Phone 15767745273
Organization name The First Affiliated Hospital of SunYat-sen University
Street address Zhong shan Second Road
City Guang Zhou
State/province Guang dong Province
ZIP/Postal code 510000
Country China
 
Platform ID GPL24676
Series (1)
GSE265794 TGFBR3 inhibits the aggressive of papillary thyroid cancer by inhibiting PI3K/AKT pathway and EMT
Relations
BioSample SAMN41074596
SRA SRX24360760

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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