Journal: bioRxiv
Article Title: An optimised protocol for quality control of gene therapy vectors using Nanopore direct RNA sequencing
doi: 10.1101/2023.12.03.569756
Figure Lengend Snippet: Plot showing the sequencing coverage (green), location of the 3’ ends of reads (orange) and splicing patterns (purple lines connecting splice donors and acceptors) for two Wiskott-Aldrich Syndrome vectors sequenced by Nanopore direct RNA technology (A) WAS LV5 sequencing data (contains a single A to T mutation in the potential cryptic polyA site). (B) Reference sequence of the Wiskott-Aldrich Syndrome vector, with various features annotated.
Article Snippet: Artificial polyadenylation was carried out based on Crabtree et al. ( ) Reactions containing 15 μL of RNA (500 ng), 2 μL of polyA buffer, 2 μL of 10 mM ATP (NEB P0756S), 1 μL of E. coli Poly(A) Polymerase (NEB M0276S) and 0.5 μL of Murine RNase Inhibitor (NEB M0314S) were incubated for 30 minutes at 37°C, 20 minutes at 65°C and then 5 minutes at 98°C before being placed on ice.
Techniques: Sequencing, Mutagenesis, Plasmid Preparation