Total RNA was extracted from each brushing sample according to the manufacturer’s instructions using the miRNA easy Mini Kit (QIAGEN, Hilden, Germany). Quantification was performed using the QuantiFluor RNA System (Promega, Madison, WI), and 50 ng of RNA was used as input to the TruSeq RNA Access Library Prep procedure (Illumina, San Diego, CA), which enriches for the coding transcriptome. Libraries meeting quality control criteria for amplification yields were sequenced using NextSeq 500 instruments (2 × 75 bp paired-end reads) with the High Output Kit (Illumina, San Diego, CA).
Raw sequencing (FASTQ) files were aligned to the Human Reference assembly 37 (Genome Reference Consortium) using the STAR RNA-seq aligner software [18 (link)]. Samples and sequencing runs that met pre-specified criteria for the number and quality of reads, as well as genomic representation and read depth, were used for downstream analysis. Based on annotated Ensembl genes, uniquely mapped reads were summarized using HTSeq [19 (link)]. The sequencing data was further filtered and normalized as described in [14 (link)].
Raw sequencing (FASTQ) files were aligned to the Human Reference assembly 37 (Genome Reference Consortium) using the STAR RNA-seq aligner software [18 (link)]. Samples and sequencing runs that met pre-specified criteria for the number and quality of reads, as well as genomic representation and read depth, were used for downstream analysis. Based on annotated Ensembl genes, uniquely mapped reads were summarized using HTSeq [19 (link)]. The sequencing data was further filtered and normalized as described in [14 (link)].
Free full text: Click here
