4.3. Bioinformatic Analysis and Hypoxic Score Calculation

The mRNA from the 786-O parental cell line as well as the derived cells overexpressed with wildtype or L169P human VHL was extracted and sequenced for mRNA expression profiles at UCLA Technology Center for Genomics & Bioinformatics (TCGB) core.

Libraries were prepared with a KAPA Stranded mRNA-Seq kit. The workflow consisted of mRNA enrichment and fragmentation, first-strand cDNA synthesis using random priming followed by second-strand synthesis converting cDNA:RNA hybrid to double-stranded cDNA (dscDNA), and it incorporated dUTP into the second cDNA strand. cDNA generation was followed by end repairing to generate blunt ends, A-tailing, adaptor ligation and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on an Illumina NovaSeq 6000 device for a PE 2 × 100 run. Data quality checking was carried out on an Illumina SAV device. Demultiplexing was performed with the Illumina Bcl2fastq v2.19.1.403 software.

The Partek flow software (Partek^® Flow^® software, version 7.0 Copyright©; 2019 Partek Inc., St. Louis, MO, USA) was used for the data analysis. The alignment and generation of the raw read counts per gene were performed using STAR-2.7.9a [38,39] with human reference genome GRCh38 genome and were annotated with Ensembl 104 GTF. Normalization of transcript counts was performed using CPM (counts per million) normalization followed by adding 0.0001. The Partek GSA algorithm was used for differential gene expression, with p < 0.05 and FDR < 0.05, and 2Fold was used for cutoffs to obtain lists of significantly expressed genes from the sample comparisons.

The hypoxia scores of the three cell lines were calculated using the mRNA-based signatures developed by Ragnum [21], Buffa [22] and Winter [23]. The raw read counts from the three cell lines were normalized with the TCGA database level 1 data using the DESeq2 package v1.38.3 accessed by 1 June 2023, and the hypoxia scores were calculated as described in Bhandari et al. [29].