Bulk RNA-Seq of OSCC patient: Buccal mucosa
Total RNA was used for library preparation using TruSeq Stranded total RNA library (illumina Inc., San Diego, CA, USA) following the manufacturers protocol. Briefly, the total RNA was subjected to rRNA depletion, without fragmentation. The first strand cDNA synthesis was performed using random hexamers and SuperScript II Reverse Transcriptase. The second cDNA strand synthesis was performed by substitution of dTTP with dUTP. The double stranded cDNA was end-repaired and adenylated. Barcoded DNA adapters were ligated to both ends of the double stranded cDNA and then amplified. The librarys quality was checked using an Agilent 2100 Bioanalyzer system and quantified using Qubit system. The sequenced raw data of primary cells derived from patient tumor sample and monolayer of cells generated from tumors were processed to obtain high-quality reads using fastp to remove the adapter sequences and reads with more than 10% quality threshold (QV)<20 Phred score. After removing the adapter and low-quality sequences from the raw data, high-quality reads were obtained. The RNA-Seq paired-end sequencing library was prepared from high-quality reads using Illumina TruSeq. After obtaining the Qubit concentration for the library, the paired-end Illumina library was loaded onto NextSeq500 for sequencing. Pair-end FASTQ files were subsequently aligned to the GRCh38 reference genome using Spliced Transcripts Alignment to a Reference (STAR) expression count. STAR has shown high accuracy as it is substantiated by an algorithm that functions by seed searching followed by clustering, stitching, and scoring.