Small RNA Sequencing Protocol

Total RNA for each biological replicate and condition was size selected by denaturing polyacrylamide gel electrophoresis. Twenty micrograms of total RNA was loaded onto a 7% denaturing urea polyacrylamide gel (SequaGel; National Diagnostics) in 0.5× Tris-borate-EDTA (TBE) buffer and run at a constant power of 30 W until the bromophenol blue bands reached the bottom of the gel. The gel was stained with SYBR Gold and visualized on a blue light box, and bands in the 50 to 500 nucleotide range, as indicated by the RNA Century Marker plus ladder (Thermo Fisher), were excised. Small RNAs (sRNA) were eluted by rotating overnight in 1.2 ml 0.3 M NaCl and were ethanol precipitated and DNase I (NEB) treated (37°C for 2 h) as previously described (69 (link)). Strand-specific libraries were prepared using the SMART-seq Ultralow RNA input kit (TaKaRa), and insert sizes were checked with the Bioanalyzer RNA pico kit (Agilent). Paired-end sequencing (2 × 150 bp) was carried out on the Illumina HiSeq 2500 platform at the Johns Hopkins University Genetic Resources Core Facility (GRCF).