Mung bean nuclease buffer

100 ng of RainDance PCR products were mixed with 2.5 µl of 10× mung-bean nuclease buffer (New England Biolabs), 2 µl of 10 units/µl mung-bean nuclease (New England Biolabs) and nuclease-free water to bring to a final reaction volume of 25 µl. The digestion reaction was incubated at 30°C for 30 min. After nuclease treatment, DNA was purified using 37.5 µl of Agencourt Ampure XP beads (Beckman Coulter) following manufacturers' instructions and eluted in 42.5 µl of nuclease-free water. Untreated samples consisting of 100ng aliquots of captured DNA from the same batch of RainDance enrichment served as paired controls.

A 40-nt rRNA fragment was purified similarly to previously described (31 (link)). Briefly, 3 to 4 μg of a biotinylated DNA probe designed to bind to the target rRNA region was combined with 33 μg of total RNA in 3.33x hybridization buffer (250 mM HEPES pH 7, 500 mM KCl) in a total volume of 150 μl. The mixture was incubated at 90 °C for 7 min and then allowed to slowly cool to room temperature over 3.5 h to allow hybridization. Single-stranded RNA and DNA were digested by adding 0.5 μg RNase A (Thermo Scientific) to the mixture and incubating at 37 °C for 30 min, then 30 units of Mung Bean Nuclease were added along with 10x Mung Bean Nuclease buffer (NEB) to a final concentration of 1x, followed by a 30 min incubation at 37 °C. Streptavidin T1 beads (20 μl per sample; Invitrogen) were washed three times in 1 ml 2.5x IP buffer (375 mM NaCl, 125 mM Tris pH 7.9, 0.25% NP-40), then resuspended in 100 μl 2.5x IP buffer. The beads were combined with the 150 μl RNA-DNA hybridization mix and rotated at 4C for 1 h. After rotation, the beads were washed three times in 1x IP buffer (150 mM NaCl, 50 mM Tris pH 7.9, 0.1% NP-40), twice in nuclease-free water, and resuspended in 30 μl nuclease-free water. The resuspended beads were heated at 70 °C for 5 min to denature the RNA from the probes and quickly placed on ice before placing on a magnet to collect the RNA-containing supernatant.