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Status |
Public on Apr 15, 2024 |
Title |
TTA-1, DMSO, rep2 |
Sample type |
SRA |
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Source name |
TTA-1
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Organism |
Homo sapiens |
Characteristics |
cell line: TTA-1 cell type: Anaplastic thyroid carcinoma genotype: BRAF WT treatment: DMSO
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Treatment protocol |
Cells were seeded on 6-well plates and treated with GDC-0994 (1μM) or DMSO for 24 hours.
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Growth protocol |
KHM-5M was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and TTA-1 was cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS).All cells were incubated under the condition of 37 °C and 5% CO2.
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Extracted molecule |
total RNA |
Extraction protocol |
Total amounts and integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies,, CA, USA) Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA by using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase, then use RNaseH to degrade the RNA. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and dNTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR amplification, the PCR product was purified by AMPure XP beads, and the library was finally obtained. In order to ensure the quality of the library, the library needs to be tested. After the construction of the library, the library was initially quantified by Qubit2.0 Fluorometer, then diluted to 1.5ng/ul, and the insert size of the library is detected by Agilent 2100 bioanalyzer. After insert size meets the expectation, qRT-PCR is used to accurately quantify the effective concentration of the library (the effective concentration of the library is higher than that of 2nM) to ensure the quality of the library.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
TTA1_DM2
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Data processing |
Clustering and sequen:After the library is qualified, the different libraries are pooling according to the effective concentration and the target amount of data off the machine, then being sequenced by the Illumina NovaSeq 6000. The end reading of 150bp pairing is generated. The basic principle of sequencing is to synthesize and sequence at the same time (Sequencing by Synthesis). Four fluorescent labeled dNTP, DNA polymerase and splice primers were added to the sequenced flowcell and amplified. When the sequence cluster extends the complementary chain, each dNTP labeled by fluorescence can release the corresponding fluorescence. The sequencer captures the fluorescence signal and converts the optical signal into the sequencing peak by computer software, so as to obtain the sequence information of the fragment to be tested. Quality control:The image data measured by the high-throughput sequencer are converted into sequence data (reads) by CASAVA base recognition. Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing N base and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality Reads mapping to the reference genome:Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 (v2.0.5) and paired-end clean reads were aligned to the reference genome using Hisat2 (v2.0.5). We selected Hisat2 as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools Quantification of gene expression level:featureCounts (v1.5.0-p3) was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression level Assembly: GRCh38, NCBI Supplementary files format and content: tab-delimited text file includes raw counts for each Sample Supplementary files format and content: tab-delimited text files include FPKM values for each Sample
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Submission date |
Apr 15, 2024 |
Last update date |
Apr 15, 2024 |
Contact name |
Yulu Chen |
E-mail(s) |
chenylu8@mail2.sysu.edu.cn
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Phone |
18374845932
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Organization name |
The First Affiliated Hospital of Sun Yat-sen University
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Department |
Precision Medicine Research Institute
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Lab |
Rengyun Liu Lab
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Street address |
No. 58 Zhongshan Second Road
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City |
guangzhou |
State/province |
guangdongsheng |
ZIP/Postal code |
510000 |
Country |
China |
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Platform ID |
GPL24676 |
Series (1) |
GSE264049 |
RNA-seq data of BRAF wild-type and BRAF mutant tumor cells treated with the ERK inhibitor GDC-0994 |
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Relations |
BioSample |
SAMN40975071 |
SRA |
SRX24268087 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
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