GSM4039266: S18_DM_RPAD2; Mus musculus; RNA-Seq
Total RNA from cultured cells was isolated using the miRNeasy Mini Kit (#217004, Qiagen Inc.) following the manufacturer's protocol. For the RPAD samples, a 20-µl reaction was set with 2 µg of Hela RNA, 20 units of RiboLock RNase inhibitor (Thermo Fisher Scientific), 1× RNase R buffer and 20 units of RNase R (RNR07250, Epicentre), and incubated for 30 min at 37°C. The RNA from control and RNase R-treated samples were isolated using miRNeasy Mini Kit following the manufacturer's instructions. The RNase R treated sample was treated with PolyA polymerase to add polyA tail to linear RNAs followed by incubation with Oligo-dT Dynabeads from Poly(A)Purist™ MAG Kit (AM1922) and the supernatant was collected for RNA isolation. The miRNeasy Mini Kit was used to prepare RNA from the control and RNase R-polyadenylation and poly(A)+ RNA depletion (RPAD) samples. After isolation of total RNA and RPAD-prepared RNA using miRNeasy Mini Kit, ribosomal (r)RNA was removed by rRNA Depletion Nano kit (Qiagen), whereupon cDNA was prepared and amplified using the Ovation RNA-Seq System V2 (NuGEN) kit following the manufacturer's instructions. The amplified cDNA was fragmented using a Bioruptor (Diagenode) followed by ligation of adaptors using TruSeq ChiP Sample Preparation kit (Illumina, San Diego, CA) and DNA fragments ranging 300-350 bp were isolated from a 2.5% agarose gel and subjected to 17 cycles of PCR amplification. A Bioanalyzer 2100 instrument was used to analyze the quality of the cDNA libraries, which were sequenced using an Illumina HiSeq2500 instrument.