5'XP Small RNA-seq: A simple sequencing method that identifies transcripts with and without 5' phosphorylation in single libraries from low input samples

Small RNA (sRNA) sequencing has been critical for our understanding of transcriptional regulation in most biological processes. Nonetheless, the varying biochemical properties of sRNA--particularly the 5' nucleotide modification--makes many sRNA subspecies incompatible with common protocols for sRNA sequencing. Here we describe 5XP-seq that outlines a novel strategy on how to overcome this limitation. By specifically tagging 5'-phosphorylated sRNA (5'P sRNA)--enriched with miRNA and piRNA--we successfully discriminate these sRNA from all other sRNA sub-species. Our results illustrate the importance of more holistic approaches--where 5' insensitive library strategies are combined with 5' sensitive tagging--to solve the growing publication bias of "easy-to-catch" sRNA.The data submitted here comes from drosophila early embryos (0.5-2.5 h old), and contains 3 different library preparation methods: 5P-seq, 5X-seq and 5XP-seq. 5P-seq is almost identical to NEBNext Multiplex Small RNA Library prep kit for Illumina (New England Biolabs) where the 5' adaptor is ligated to RNA using a 5'-P dependent ligase. 5X-seq used a protocol insensitive to 5' nucleotide modifications, where the 5' adaptor was ligated to the corresponding 3' end of cDNA after reverse transcription. 5XP-seq combined these two methods by tagging the 5'-P with a short oligo using the 5'-P dependent ligase, while the 5' adaptor was later ligated to the cDNA.