Long Nuclear RNA-seq Library Preparation

RNA was extracted from 1 million sorted and washed nuclei using the standard procedure (Jänes et al. 2018 (link)). A minimum of 20 ng of total nuclear RNA was used to make long nuclear RNA-seq libraries. Long nuclear RNA (>200 nt) was isolated using Zymo Clean and Concentrate columns (R1013); rRNA was removed using the Ribo-Zero rRNA removal kit (MRZH11124); and stranded libraries were prepared with the NEBNext ultra directional RNA library prep kit (E7420S). Long nuclear RNA-seq libraries were generated from two biological replicates for each tissue and were sequenced in paired-end mode. We observed that all tissue-specific libraries have noticeable background for abundant tissue-specific mRNAs (e.g., muscle myosin unc-54). This appears to be due at least in part by contamination by whole-animal cytoplasmic RNA released during nuclear isolation, as the RNA in the unexpected tissue is predominantly spliced.