RNA-seq of C. briggsae HK104 mixed-stage embryos
For AF16 and HK104 mixed-stage embryo RNA-seq, three full, non-starved 9 cm plates of the appropriate worm strain were first bleached to isolate only the embryos, then total RNA was extracted from them using a TRIzol extraction method based on a previously published protocol (75) with the following modifications - chloroform was used instead of bromochloropropane, and phase-lock tubes were used during TRIzol/chloroform steps. DNA was removed from the samples with a DNase I digestion (30 mins at 37C), then the enzyme was inactivated with 2.5 L of 25 mM EDTA and a 10 mins incubation at 65C. 100 ng of purified RNA was used as input for the TruSeq Stranded mRNA kit (Illumina; cat. #20020595) and libraries were prepared according to manufacturer instructions. Libraries were then checked on a Fragment Analyzer and sequenced on a 2x75 paired-end NextSeq550 lane. All samples were run in biological triplicate.