Long read sequencing of reporter plasmids and RNA
Long read sequencing of reporter plasmids and RNA
Samples were derived from plasmid DNA, or from whole cells transfected for 16 hours with the plasmid pool. In the case of the transfected cells, cells were grown in DMEM+Glutamax+10% FBS and seeded such that they achieved 70% confluency on the morning of the experiment, with each sample being derived from 2 wells of a 6 well plate. In the case of the transfected cells, cells were transfected with the plasmid pool using Lipofectamine 3000 according to manufacturer instructions. In the case of the transfected cells, RNA extractions were performed from cell pellets using the Maxwell RSC simplyRNA kit (Promega) using a Maxwell RSC Instrument (Promega). Targeted PCR of the whole transcript, followed by Nanopore adapter ligation. In the case of the RNA sequencing, we performed oligo-dT based RT using Superscript IV