title: JCVI-syn1.0 ONT Replicate 2
design: Cellular RNA was extraced using an adapted phenol-chloroform extraction protocol and the ThermoFisher TRIzolTM Reagent user guide. 1mL of TRIzol added to sample and centrifugation was used to seperate the RNA containing aqueous phase. RNA pellets were disolved and RNA concentration was measured using Qubit RNA HS fluorometer assay. rRNA was depleted from the sample using the NEBNext rRNA Depletion Kit for bacteria. 2 L of NEBNext Bacterial rRNA Depletion Solution and 2 L of NEBNext Probe Hybridization Buffer was added to a solution of 1 g of RNA and 11 L of RNase-free water. rRNA was extracted with bead cleanup with Agencourt RNAClean XP beads. Poly(A) tailing was performed using 2 l of 10X E. coli Poly(A) Polymerase Reaction Buffer, 2 L of ATP and 1 L of the E.coli Poly(A) Polymerase (New England Biolabs). RNA was normalized to 140 ng. Libraries for Oxford Nanopore sequencing were prepared using the ONT Direct RNA Sequencing Kit (SQK-RNA002) with slight modifications, 0.5 L of the RT adapter enzyme (reduced from 1 L) and 4 L of the RMX RNA adapter (reduced from 6 L). Sequencing was done using R9.4 flow cells on the ONT GridION platform.