Ribosomal RNA Depletion and mRNA Sequencing

Ribosomal RNAs were depleted using the RNase H method^{108 (link)}. 3 μg total RNA were incubated with antisense DNA oligonucleotides targeting C. elegans and E. coli ribosomal RNAs, then treated with Hybridase Thermostable RNase H (Biosearch Technologies) at 45 °C for 30 min. The rRNA-depleted samples were treated with Turbo DNase (Thermo Fisher Scientific) at 37 °C for 30 min. RNAs longer than 200 nts were enriched using RNA Clean & Concentrator-5 (Zymo Research). mRNA sequencing libraries were constructed using Ultra II Directional RNA Library Prep Kit (NEB), according to the manufacturer’s instructions. The library samples with unique barcodes were pooled and sequenced at the Illumina HiSeq 4000 platform (PE 150 bp).