GSM5707497: TS543_DMSO_1; Homo sapiens; RNA-Seq
Tree days after treatment, cells were harvested and total RNA were extracted with RNeasy® Micro Kit (Qiagen). A modified NEBNext RNA Ultra protocol is used to prepare the libraries for mRNA sequencing. The first step is selectively purifying the poly-A containing mRNA molecules using oligo-dT attached magnetic beads. Following purification, the mRNA is fragmented using divalent cations under elevated temperature. Next, the cDNA is synthesized using Reverse transcriptase and random hexamers in a first strand synthesis reaction. Subsequently, the cDNA is converted to double stranded cDNA in a DNA polymerase based reaction. These DNA molecules are then subjected to a series of enzymatic steps for end repair, A-Tailing and ligation with NEB adapters. Finally, these adapter ligated molecules are enriched in a limited cycle PCR with indexed primers and purified using AMPure beads to create final cDNA library for sequencing.