Small RNA Sequencing Protocol for Transgene Analysis

Small RNAs (17–25 nt size) from 20 $\mathsf{\mu }$ g total RNA were size selected by gel extraction using a 17.5% urea acrylamid mini-gel, visualized with SYBR-Gold (Life-Technologies, Darmstadt, Germany). Gel slices were cut and RNA was eluted by overnight incubation in 3 Vol. using 0.3M NaCl at 4 ${}^{\circ }$ C, RNA was precipitated using 3 Vol. Ethanol (100%) and Glycogen (70 ng/ $\mathsf{\mu }$ L) and afterwards subjected to library preparation using the NEBNext small RNA library prep Kit (New England Biolabs, Frankfurt a.M., Germany), according to the manufacturer’s instructions (3 ${}^{\prime }$ -adapter ligation extended to 18h at 16 ${}^{\circ }$ C). Sequencing was done on the Illumina HiSEQ 2500 platform (Illumina, San Diego, CA, USA) using the RAPID mode and 30 nt read length. Reads (1.9; 6.7; 5.5 million Mac mapping reads, respectively) were de-multiplexed and adapter sequences were trimmed using Trim Galore [20 ] that uses Cutadapt [21 (link)] with a stringency cutoff of 10. SmallRNA read alignments to transgenes and normalization of reads were carried out precisely as described in [17 (link)]. The complete analysis was done using the RAPID pipeline [22 ].