Candida auris ribosomal RNA
For rRNA sequencing, RNA from 10 g of ribosomes were purified on Monarch RNA Cleanup 10 g columns (New England Biolabs, cat, T2030L). 1 g RNA was then mixed with 1 l 50 M random hexamers, 2 l 10 mM dNTPs, and 4 l of First Strand Buffer 5X [250 mM Tris-HCl, pH 8.3; 375 mM KCl; 15 mM MgCl2] in 17 l final, and incubated at 94C for 6 minutes to fragment the RNA. Fragmented RNA was then quickly chilled on ice for 2 min, before adding 2 l 0.1 M DTT, 1 l TGIRT-III reverse transcriptase (InGex) and 1 l SUPERaseIn RNase Inhibitor (ThermoFisher Scientific, cat. AM2694). RNA was reverse transcribed for 10 min at 25C, 30 min at 57C, and 30 min at 60C, after which the reverse transcriptase was heat-inactivated by incubating at 75C for 15 min. RNA was then used as input for the NEBNext UltraTM II Non-Directional RNA Second Strand Synthesis Module (New England Biolabs, cat. E6111S), and second strand synthesis was performed as per manufacturer instructions. The resulting dsDNA was then used as input for the NEBNext UltraTM II DNA Library Prep Kit for Illumina kit (New England Biolabs, cat. E7645S) as per manufacturer instructions.