RNA-Seq Analysis of Calotropis Gigantea leaf extract treated with Hela, MCF7 and A549 cancer cell lines.

Transcriptome analysis is the promising technology to identify the differentially expressed genes to predict functional drug targets. In the present study is based on the treatment of calotropis gigantea leaf extracts as crude sample with different cancer cell lines MCF7 HeLa and A549 to predict the gene regulations. The treatment is carried out with different concentrations of IC50 values such as MCF7 43.65 microgram per ml A549 27.32 microgram per ml and HeLa 117.92 microgram per ml with 24hours of incubation period and the resultant samples were used for gene expression to predict drug targets. Here, we isolated Total RNA using Quick-RNA Miniprep Plus kit (ZYMO Research). The RNA-seq paired end sequencing libraries were prepared using Illumina TruSeq Stranded mRNA sample Prep kit and the PE illumina libraries were loaded onto NextSeq 500 for cluster generation and sequencing. The kit reagents were used in binding of samples to complementary adapter oligos on paired-end flow cell. The adapters were designed to allow selective cleavage of the forward strands after re-synthesis of the reverse strand during sequencing. The copied reverse strand was then used to sequence from the opposite end of the fragment. The mean of the libraries fragment size distributions are 457bp, 443bp and 441bp for HeLa, MCF7 and A549 tests, respectively. The libraries were sequenced on NextSeq 500 using 2 x 75 bp chemistry. Bioinformatics analysis were performed based on quality interpretation by FASTQC, Reference based alignment using Bowtie and TopHat to for sequence annotation and alignment. Cufflinks is used to predict differential expressed genes; R is used for statistical interpretation of functional genes as potential drug targets.