smallRNA-seq of pid-4; pid-5; mCherry::21U sensor (RNAe) - rep 2 RppH treated
For each sample, 1 g of RNA was incubated for 1 hour at 37 C with 5 units of RppH and 10X NEB Buffer 2. After dephosphorylation, 500 mM EDTA was added and samples were incubated for 5 minutes at 65C to stop the RppH treatment and RNA was purified with sodium chloride/isopropanol precipitation. After purification, the recovered total RNA was used for library preparation. Small RNAs (15-30nt) were enriched by performing size selection of the RNA prior to library preparation. NGS library prep was performed with NEBNexts Small RNA Library Prep Kit for Illumina following instructions of manual, with a modification of the adaptors, for which custom made random barcodes for both 3 SR Adaptor and 5 SR Adaptor were used (HISS Diagnostics GmbH, 5rApprnrnrnrnAGATCGGAAGAGCACACGTCT-NH2-3, and 5'rGrUrUrCrArGrArGrUrUrCrUrArCrArGrUrCrCrGrArCrGrArUrCrnrnrnrn-3, respectively). Libraries were prepared with a starting volume of 6l and amplified in 14 PCR cycles. Libraries were profiled in a High Sensitivity DNA on a 2100 Bioanalyzer (Agilent technologies) and quantified using the Qubit dsDNA HS Assay Kit, in a Qubit 2.0 Fluorometer (Life technologies).