title: GSM7882563: PltBS35A typhoid toxin treated-1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNAs from the cells were isolated using RNeasy purification kits (QIAGEN) following the manufacturer's instructions. The concentration of RNA was measured by using Qubit RNA BR assay kit with Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, United States). RNA integrity Number (RIN) of RNA was assessed by using an RNA Nano 6000 assay kit and Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, United States). 100 ng of RNA with RIN ≧ 8 was used for library preparation with illumina stranded mRNA library prep Kit (Illumina, San Diego, CA , United States). Briefly, messenger RNA (mRNA) with polyA tails was isolated by using oligo(dT) magnetic beads. Purified mRNA was fragmented into short segments and complementary DNA (cDNA) was synthesized with random hexamer primers. To achieve strand specificity, the deoxyuridine triphosphate (dUTP) was used for the replace of deoxythymidine triphosphate (dTTP) while performing second-strand cDNA synthesis. cDNA was clean up with AMPure XP beads (Beckman Coulter, Indianapolis, Indiana, United States) and followed by end repair, polyadenylation, and Illumina sequencing adapter ligation. After proper PCR amplification, final library was purified and paired-end 150 bp sequencing was performed with Illumina Novaseq to achieve 20 M reads per sample.