Computational protocol: HIF-1-mediated suppression of mitochondria electron transport chain function confers resistance to lidocaine-induced cell death

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[…] FASTQ files for RCC4-EV cells (SRR1554431 and SRR1554986) and RCC4-VHL cells (SRR1554988 and SRR155499) were obtained from the Sequence Read Archive (https://trace.ddbj.nig.ac.jp/dra/index_e.html). The quality of sequence data was evaluated by FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) after the trimming process by fastx_toolkit v 0.0.14 (http://hannonlab.cshl.edu/fastx_toolkit/). The human reference sequence file (hs37d5.fa) was downloaded from the 1000 genome ftp site (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/phase2_reference_assembly_sequence/), and the annotated general feature format (gff) file was downloaded from the Illumina iGenome ftp site (ftp://igenome:[email protected]/Homo_sapiens/NCBI/build37.2/). The human genome index was constructed with bowtie-build in Bowtie v.2.2.9. The fastq files were aligned to the reference genomic sequence by TopHat v.2.1.1 with default parameters. Bowtie2 v2.2.9 and Samtools v.1.3.1 was used with the TopHat program. Estimation of transcript abundance was calculated, and the count values were normalized to the upper quartile of the fragments per kilobase of transcript per million fragments mapped reads (FPKM) using Cufflinks (cuffdiff) v2.1.1.Metascape (http://metascape.org/) was used for the gene set enrichment analysis. A gene list for metascape analysis was generated using the output from the cuffdiff program, in which 72 genes judged as ‘significantly differentially expressed’ (p < 0.05) in cuffdiff output (gene_exp. diff, Supplementary Dataset ) were contained (Supplementary Fig. ).Genes containing Gene Ontology annotations (‘canonical glycolysis’ (GO:0061621) and ‘hypoxia-inducible factor-1alpha signaling pathway’ (GO:0097411)) were extracted using Ensembl Biomart and sorted by corresponding values (common logarithms of ([FPKM of RCC4-EV] + 1)/([FPKM of RCC4-VHL] + 1)) calculated from the same cuffdiff output file (Supplementary Fig. ). The integer one was added to all FPKM values because we cannot calculate the logarithm of 0. The histogram was generated using TIBCO Spotfire Desktop v7.6.0 with TIBCO Spotfire’s “Better World” program license (TIBCO Spotfire, Inc., Palo Alto, CA, USA) (http://spotfire.tibco.com/better-world-donation-program/). […]

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