GSM5218536: siRNA against firefly luciferase, replicate 3; Homo sapiens; RNA-Seq
After 48 hours, cells were collected and subjected to further analysis. Three replicates were taken for each condition. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads and then fragmented randomly by addition of fragmentation buffer. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Double-stranded cDNA was purified using AMPure XP beads. Remaining overhangs of the purified double-stranded cDNA are converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure is ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Finally, the final library was prepared by PCR amplification and purification of PCR products by AMPure XP beads.  Paired-end 150bp reads were sequenced on an Illumina NovaSeq and base-called with CASAVA.