Instrument: NextSeq 500
Strategy: ncRNA-Seq
Source: TRANSCRIPTOMIC
Selection: size fractionation
Layout: SINGLE
Construction protocol: Meiocyte or tapetum cells was isolated using the same method as for BS-seq.Total RNA was then extracted from isolated pure meiocyte or tapetum cells using Direct-zol RNA Kit (Zymo, #R2061). sRNA library was constructed using the RealSeq-Biofluids NGS Library Preparation Kit (Biocat,600-00048-SOM).To further enrich the small RNA fractions of the library and remove the potential adaptor dimers, the generated sRNA library was separated by electrophoresis with a 6% TBE gel (Novex, #EC6265BOX). The region containing DNA bands from 147 bp to 157 bp long (corresponding to 20-30 nt sRNA size) was excised with a knife and then resolved in elution buffer (0.5 M ammonium acetate, 10 mM magnesium sulfate, 1 mM EDTA (pH 8.0), 0.1% SDS) for 4 hours at 37°C. The remaining fragments of polyacrylamide were removed by passing the supernatant through a disposable plastic column. Two volumes of ethanol at 4°C were added to the solution and then stored on ice for 30 min. DNA was recovered by centrifugation for 10 min at 4°C in one microcentrifuge tube. Finally, the DNA was washed with 70% ethanol and resolved in Tris-EDTA (TE) buffer (pH 7.6).