title: RNA seq  of A549 human lung cancer cells
design: Prepared libraries were sequenced on Illumina HiSeqX to generate 60M, 2x150bp reads/sample.  Initially the raw fastq files of all samples were quality checked using FastQC tool Version 0.11.9. The base quality score distribution, sequence quality score distribution, average base content per read, GC distribution in the reads, PCR amplification issue, over-represented sequences were checked for the samples. Based on the quality report of fastq files, sequence read were trimmed where necessary to only retain high quality sequence for further analysis. In addition, the low-quality sequence reads were excluded from the analysis. The adapter trimming was performed using Trimmomatic (v-0.36).  As a part of pre-processing step, we are removing the unwanted sequences like mitochondrial genome sequences, ribosomal RNAs, transfer RNAs, adapter sequences and other non-coding RNAs using Bowtie2 version 2.2.4.  The contamination filtered paired-end reads were mapped to the reference human genome (hg19) and GFF annotation file using HISAT2 tool (2.1.0) with default parameters. The aligned reads are used for estimating expression of the genes. The raw read counts were estimated using Feature Count (1.5.2). Read count data were normalized using negative binomial approach using DESeq2 package available in R-software 4.1.0 version.