RNA-seq of Xenopus laevis (eggs) x tropicalis (sperm) hybrids
Embryos were washed quickly with ~1.5 ml cold RNase-free embryo wash buffer (EWB; 10 mM K-HEPES, pH 7.7, 100mM KCl, 1 mM MgCl2, 0.1 mM CaCl2, 50mM sucrose). Immediately after removal of EWB, 500 l of Tripure was added and tubes vortexed for 30 seconds to homogenize the embryo until no visible pieces remained. 400 l additional Tripure was added and samples flash frozen in liquid nitrogen and stored at -80C until RNA extraction. Embryo samples in Tripure reagent were thawed at room temperature and placed on ice. ERCC spike-in RNAs (4456653; ThermoFisher Scientific) were added at a 1:40 dilution of stock at 1l per embryo (5 embryos and 5 l diluted ERCC RNA per sample) to the samples in Tripure and mixed gently by inversion followed by 2 seconds of vortexing. RNA was extracted with 200 l chloroform using standard procedures and resuspended in 20 l DEPC-treated H20. RNA concentrations were measured by nanodrop and total RNA integrity assayed using an Agilent Bioanalyzer. All RNAs had a RIN (RNA integrity number) greater than 9.0. RNA (10 g) was treated with DNase (TURBO DNase; Ambion) for 1 hour at room temperature followed by isolation with a minElute RNA Cleanup Kit (Qiagen). 5 g DNase-treated RNA was used as input for ribosomal depletion using the Epicentre (Illumina) Ribo-Zero ribosomal depletion kit (Cat# SCL24H). Ribosomal RNAs were depleted and RNA quality was assessed post-depletion using a bioanalyzer to ensure loss of 18S and 28S rRNA. rRNA-depleted RNA was then purified using Ampure SPRI beads with a 1.8X bead to sample ratio. RNA was converted to cDNA libraries using the stand-specific Script-seq V2 RNA-seq kit (Illumina) according to the manufacturers instructions. 14 PCR cycles were used to amplify libraries and samples were indexed for multiplexing. To reduce lane effects on individual samples within a replicate, indexed libraries from each time point and embryo treatment condition (hybrid and cybrid) were pooled into a single replicate sample.