NextSeq 500 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 Pan-Prokaryote riboPOOL kit (siTOOLs). 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 and purified by phenol / chloroform extraction. RNA was sheared in a microTUBE AFA Fiber Pre-Slit Snap-Cap (130 ï¿½L) for 140 s in a Covaris M220 Focused-ultrasonicator, purified using RCC-5, and treated with T4 Polynucleotide Kinase (New England Biolabs) for end repair. After purification using RCC-5, the library was prepared using the NEXTflex Small RNA-Seq Kit v3 (Bioo Scientific) according to the manufacturer's protocol with 18 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 NextSeq 500.