GSM2773220: cytoRNA-2.0_2nd 2.0 ng/ul RNase A; Homo sapiens; OTHER
cytoplasmic RNA was purified with hypotonic buffer, followed by douncing 50 times. RNA was purified using Qiagen RNeasy Mini Kit. RNAs were first incubated with recombinant  MDA5 proteins, then challenged by two concentrations of RNaseA. The reaction was terminated by TRIzol. RNA was recovered and rRNA was depleted using NEBNext rRNA depletion kit. cDNA library was constructed using Clontech Smarter Stranded RNA-seq Kit. Mock-treated RNAs (cytoRNA-0.0) were fragmented by heating at 94C for 3min, RNaseA-digested RNAs (cytoRNA-0.5 and cytoRNA-2.0) were fragmented by heating at 94C for 1min without First-strand Buffer. PCR cycles for cytoRNA-0.0 was 15, for cytoRNA-0.5 and -2.0 were 18. The Ampure Beads:DNA ratio was 0.8:1 (volume:volume) for DNA purification.