title: degradome-seq from w1 ovary
design: -2 days old w1 flies were collected and fed with yeast. Ovaries were dissected after 3 days. Total RNA was extracted with mirVana kit (Ambion). Two rounds of rRNA deletion was done with riboZero (epibio) kits, followed by turbo DNase treatment (Ambion). Five prime ligation was performed using T4 RNA ligase (ambion) at 25 degree for 3 hours. Reverse transcription using degenerate primer was performed with SuperScript III (Invitrogen). First round of PCR was used to amplify the cDNA (Q5, NEB). Then size selection 200-400 nt was performed using native PAGE. Secondary round of PCR was used to introduce Illumina Barcode (Q5, NEB), followed by size selection for 250–500 nt. Please see manuscript (will updated once published) for detailed protocol.