Illumina HiSeq 3000 paired end sequencing
Cells were lysed enzymatically using lysozyme and mutanolysin, and total RNA was extracted using TRIzol Reagent (Invitrogen) / chloroform and precipitated with isopropanol. Total RNA was treated with TURBO DNase (Invitrogen), and ribosomal RNA was depleted using the Ribo-Zero rRNA Removal Kit for bacteria (Illumina). Purification of RNA was conducted using RNA Clean & Concentrator-5 (RCC-5; Zymo Research) between each step. RNA was then treated with RppH (New England Biolabs) to convert 5' triphosphates to monophosphates, purified by phenol / chloroform extraction, and treated with  T4 Polynucleotide Kinase (New England Biolabs) for end repair. RNA was purified and separated into small and large RNA fractions using RCC-5 according to the manufacturer's protocol. The large RNA fracion (> 200 nt) was sheared in a microTUBE AFA Fiber Pre-Slit Snap-Cap (130 ï¿½L) for 140 s in a Covaris M220 Focused-ultrasonicator and purified using RCC-5. The library was prepared from the sheared large RNA fraction only using the NEXTflex Small RNA-Seq Kit v3 (Bioo Scientific) according to the manufacturer's protocol with 22 PCR cycles (until step G), purified according to step I of the NEXTflex Rapid Directional qRNA-Seq Kit protocol (Bioo Scientific) using Agencourt AMPure XP beads (Beckman Coulter), and sequenced on an Illumina HiSeq 3000.