miRNA-seq of 2S
For each RNA mixture, 500 ng of total RNA was used as input into the NEBNext Multiplex Small RNA Library Prep Kit for Illumina (New England Biolabs; Ipswich, MA) with 12 cycles of indexing and enrichment. Fragment size profiles and quantification of the final libraries were measured using a Fragment Analyzer 5300 (Agilent Technologies; Santa Clara, CA). Libraries were pooled at equimolar concentrations based on fragments within the size range of 125-160bp. The equimolar library pool was cleaned using the MinElute PCR purification Kit (QIAGEN Sciences; Germantown, MD). A PippinHT (Sage Science; Beverly, MA) with 3% agarose gel cassette was used to isolate library fragments between 125-160bp. The isolated library fragments were normalized to 0.75nM and sequenced on a NovaSeq 6000 SP flow cell with single end 101bp reads (Illumina Inc; San Diego, CA).