Low-male-3
A total amount of 1.5 g RNA per sample was used as input material for rRNA removal using the Ribo-Zero rRNA Removal Kit (Epicentre, Madison, WI, USA). Sequencing libraries were generated using NEBNextR UltraTM Directional RNA Library Prep Kit for IlluminaR (NEB, USA) following manufacturers recommendations and index codes were added to attribute sequences to each sample. Briefly, Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer  5X. First strand cDNA was synthesized using random hexamer primer and Reverse Transcriptase. Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3 ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select insert fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP Beads (Beckman Coulter, Beverly, USA). Then 3 l USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37C for 15 min before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index(X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 and qPCR.
SUB6527028
Low-male-3