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RNA sequencing of wild-type and mutant KRAS human lung cancer cell lines with of without FASN inhibition.

ABSTRACT: The goal of this study is to compare the RNA-seq derived transcriptome of mutant KRAS and wild-type KRAS lung cancer cell lines under fatty acid synthase (FASN) inhibition. We extracted total RNA with TRIzol from cells treated with 0.2 μM TVB-3664 (FASN inhibitor) or 0.2% DMSO for 4 days. One µg of total DNAse-treated RNA was used for library. cDNA was ligated to adapters and PCR amplified. Before being normalized and pooled, samples were quantified by Qubit then run on the Illumina NextSeq 500. Reads were aligned to the human hg38 reference genome. Only uniquely mapped reads were retained for further analyses. Gencode annotation for human (version v37) was used as reference alignment annotation and downstream quantification. Gene level expression was calculated using featureCount (v2.0.1) using intersection-strict mode by exon. Counts were calculated based on protein-coding genes from the annotation file. Low expressed genes were filtered using a per time-point approach with RPKM >= 0.5 in all samples in one or the other group.

ORGANISM(S): Homo sapiens

PROVIDER: GSE168782 | GEO | 2022/06/29

REPOSITORIES: GEO

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Project description:Purpose: The goals of this study are to compare the transcriptome of Arabidopsis seedlings expressing pTaF5H1:TaF5H1(expression of TaF5H1 driven by its native promoter) with the wild-type control(Col-0) under/without salt treatment, which may give us clues to whether and how cell wall composition changes affect salt tolerance. Transcriptomes profiles of 14-day-old Arabidopsis seedlings of wild-type (Col-0) and seedlings harbour pTaF5H1:TaF5H1 without and under NaCl treatment for 6 hours were generated by RNA-seq, in triplicate, using Illumina a HiSeq instrument according to manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was carried out using a 2x150 paired-end (PE) configuration; image analysis and base calling was conducted by the HiSeq Control Software (HCS) + OLB + GAPipeline-1.6 (Illumina) on the HiSeq instrument. In order to remove technical sequences, including adapters, polymerase chain reaction (PCR) primers, or fragments thereof, and quality of bases lower than 20, pass filter data of fastq format was processed by Cutadapt (V1.9.1) to be high quality clean data. For mapping, firstly, reference genome sequences and gene model annotation files of relative species were downloaded from ENSEMBL. Secondly, Hisat2 (v2.0.1) was used to index reference genome sequence. Finally, clean data were aligned to reference genome via software Hisat2 (v2.0.1). For expression analysis, in the beginning, transcripts in fasta format are converted from known gff annotation file and indexed properly. Then, with the file as a reference gene file, HTSeq (v0.6.1) estimated gene and isoform expression levels from the pair-end clean data. For differential expression analysis, DESeq2 Bioconductor package was used, a model based on the negative binomial distribution, the estimates of dispersion and logarithmic fold changes incorporate data-driven prior distributions, Padj of genes were setted <0.05 to detect differential expressed ones. Results: we mapped about 43.5 million sequence reads per sample to the Arabidopsis genome (TAIR10). Significant differential expressed genes(DEG) between samples was sreened for fold change≥2 and p-value (fdr, padj)≤0.05.Totally 7713 significant DEGs were discovered with 3430 up -regulated and 4483 down-regulated, no significant changes were detected as for the remaining 26505 genes among samples. Conclusions: Our study presents the detailed analysis of transcriptomes of Arabidopsis wild-type control(Col-0) and Arabidopsis seedlings expressing pTaF5H1:TaF5H1, with three biologic replicates, generated by RNA-seq technology. Our results show that ectopic expressing pTaF5H1:TaF5H1 in Arabidopsis changed the expression of some cell-wall related genes as well as genes related to salt stress response, inferring that changes in cell wall components may affect the salt tolerance of plants somehow.

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RNA sequencing of wild-type and mutant KRAS human lung cancer cell lines with of without FASN inhibition.

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