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Status |
Public on Jan 11, 2023 |
Title |
HUVEC_ANRIL_1 |
Sample type |
SRA |
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Source name |
ANRIL overexpression in HUVEC cell
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Organism |
Homo sapiens |
Characteristics |
cell line background: HUVEC genotype/variation: ANRIL overexpression
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Treatment protocol |
GAPDH is used as a control gene to evaluate the over-expression effect of ANRIL. cDNA was synthesized according to standard procedures. Bio-Rad S1000 was subjected to reverse transcription-polymerase chain reaction (RT-PCR) with Bestar SYBR green RT-PCR master mixture (DBI Bioscience, Shanghai, China). The concentration of each transcript was then standardized to GAPDH mRNA level through the 2- ΔΔCT method. Graphpad prism software (San Diego, California) was used for paired Student’s t-test comparison.
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Growth protocol |
HUVEC cells (8000, Sciencell) were cultured at 37 ℃ with 5% CO 2 in ECM with 5% fetal bovine serum (FBS), 100 µg/mL streptomycin, 100 U/mL penicillin and 1% growth factors. Plasmid transfection of HUVEC cells was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Transfected cells were harvested after 24h for RT-qPCR analysis.
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Extracted molecule |
polyA RNA |
Extraction protocol |
Total RNA was extracted with TRIZOL (Ambion). Total RNA was treated with RQ1 DNase (Promega) to remove DNA. The quality and quantity of the purified RNA were determined by measuring the absorbance at 260nm/280nm (A260/A280) using Nanodrop One (Thermo). RNA integrity was further verified by 1.5% agarose gel electrophoresis For each sample, 1 μg of total RNA was used for RNA-seq library preparation by KAPA Stranded mRNA-Seq Kit for Illumina® Platforms (#KK8544). Polyadenylated mRNAs were purified by VAHTS mRNA capture Beads (N401-01) and fragmented mRNAs were converted into double strand cDNA. Following end repair and A tailing, the DNAs were ligated to Diluted Roche Adaptor (KK8726). Products corresponding to 300- 500bps were amplified, purified, quantified, and stored at -80 ℃ before sequencing. The strand marked with dUTP (the 2nd cDNA strand) was not amplified, allowing strand-specific sequencing.For high-throughput sequencing, the libraries were prepared following the manufacturer's instructions and applied to Illumina Novaseq 6000 system for 150 nt paired-end sequencing
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
Illumina bcl2fastq software used for basecalling. Raw reads containing more than 2-N bases were first discarded. Then adaptors and low-quality bases were trimmed from raw sequencing reads using FASTX-Toolkit (Version 0.0.13). The short reads less than 16nt were also dropped After that, clean reads were aligned to the GRch38 genome by tophat2 (Kim, Pertea et al. 2013) allowing 4 mismatches. The R Bioconductor package edgeR was utilized to screen out the differentially expressed genes (DEGs). A false discovery rate <0.05 and fold change>2 or < 0.5 were set as the cut-off criteria for identifying DEGs The ABLas pipeline was applied to define and quantify the alternative splicing events (ASEs) and regulated alternative splicing events (RASEs) between the samples according to previously described methods Genome_build: GRCh38 Supplementary_files_format_and_content: tab-delimited text files include gene FPKM values for each Sample
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Submission date |
Feb 21, 2022 |
Last update date |
Jan 11, 2023 |
Contact name |
Dong Chen |
Organization name |
ABLife, Inc.
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Department |
Center for Genome Analysis
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Street address |
388 GaoXin 2nd Road, East Lake Hi-Tech Development Zone
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City |
Wuhan |
State/province |
Hubei |
ZIP/Postal code |
430075 |
Country |
China |
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Platform ID |
GPL24676 |
Series (1) |
GSE197115 |
ANRIL overexpression globally induces expression and alternative splicing of genes involved in inflammation in HUVECs |
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Relations |
BioSample |
SAMN26137385 |
SRA |
SRX14240876 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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