title: JCVI-syn1.0 ONT Replicate 1
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. Sample was rRNA depleted using an Invitrogen RiboMinus Bacteria 2.0 Transcriptome Isolation Kit. 2g of total JCVI-syn1.0 RNA was mixed with 100 l of RiboMinus 1X hybridization buffer and 6 L of RiboMinus Pan-Prokaryote Probe Mix. 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.