Rq1 stop solution

Fractions 1+ 2, 3+4, 16+17, and 18+19 were combined. RNA was extracted from uniform volumes of each fraction or combination of fractions. RNA extraction was performed by adding one volume of TRIzol reagent (Invitrogen), mixing until homogeneous, and incubating at room temperature for 5 min. Samples were then incubated at room temperature for another 5 min following the addition of 0.4 volumes of chloroform. After centrifugation for 15 min at 12,000×g at 4°C, the aqueous supernatant was transferred to a new tube to which 250 pg of a luciferase control RNA spike-in (luc, Promega). RNA was precipitated overnight at –20°C by the addition of 1 volume of isopropanol and 2 µl of GlycoBlue (15 mg/ml, Invitrogen). RNA was pelleted by centrifugation, washed two times with 75% ice-cold, aqueous ethanol, and allowed to dry at room temperature for ~30 min. The RNA was then resuspended in 20 µl of nuclease-free water. RNA quality and concentration were assessed by a NanoDrop UV spectrophotometer (Thermo Fisher Scientific). Genomic DNA was eliminated by incubating 10 µl of isolated RNA with 1 U of RQ1 RNase-free DNase I (Promega) for 30 min at 30°C. The DNase reaction was halted by the addition of RQ1 Stop Solution (Promega) and incubation for 10 min at 65°C.

Pellets were then thawed and resuspended in 200 µl of lysis buffer containing 30 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 15 mg/ml lysozyme, and Proteinase K (New England Biolabs). Following lysis for 10 min at room temperature, total RNA was isolated using an RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions. Yield and purity of isolated RNA were assessed by NanoDrop UV spectrophotometer (Thermo Fisher Scientific). RNA integrity was assessed by performing 1% TBE agarose gel electrophoresis with samples that had been boiled for 95°C for 5 min in RNA loading dye (New England Biolabs). Genomic DNA was eliminated by incubating 2 µg of RNA with 2 U of RQ1 RNase-free DNase I (Promega) for 30 min at 30°C. The DNase reaction was halted by the addition of RQ1 Stop Solution (Promega) and incubation for 10 min at 65°C.