Kapa hyper prep kits with riboerase

Total RNA was isolated from snap frozen oral epithelial cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The isolated RNA samples were checked for quality control using the RNA 6000 Nano Chip kit in an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries for RNA-seq were prepared from total RNA (300 ng per sample) using the Kapa Hyper Prep Kits with RiboErase (Kapa Biosystems, Wilmington, DE, USA). The workflow consisted of rRNA depletion, cDNA generation, and end repair 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 Illumina Nextseq500 for a single-end read for 75 cycles. Data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq2 v2.17 program. To rule out any potential bias, library construction and data acquisition for samples from different groups (vapers, smokers, and controls) were done in the same run, not in different batches, and in a “blind” fashion. Detailed descriptions of data processing and analysis are provided in Supplementary Data. The RNA-seq data will be deposited in the Gene Expression Omnibus database at NCBI (htttp://www.ncbi.nlm.nih.gov/geo/), and accession number will be provided as soon as it becomes available.