Small RNA and total RNA sequencing of C. elegans with mutations in the hrde-1 and emb-4 genes

Synchronised animals were grown to young adult stage at 20C on HB101 seeded NGM plates. Animals were harvested and washed 3X in M9 buffer. Settled animals were mixed with Trisure reagent, bead beaten using zirconia beads, and the RNA is extracted using chloroform. For total RNA sequencing, Illumina Ribozero kit is used to remove ribosomal RNA from 1ug of total RNA prior to library preparation. RNA sequencing libraries are prepared using NEB Next Ultra library preparation kit. Small RNA sequencing is performed by treating 5ug of total RNA with Epicentre 5’ polyphosphatase to remove the 5’ triphosphate. After treatment, RNA is purified by phenol/chloroform extraction and 1ug of RNA is used to prepare small RNA libraries using Illumina TruSeq small RNA library preparation kit. Ribosomal depleted RNA and small RNA libraries are sequenced using Illumina HiSeq 1500 platform.