C636_RepB
Intracellular RNA was extracted using RNeasy Total RNA Kit (QIAGEN, 74104). One ug of RNA was used to generate cDNA was produced using random primers and the Protoscript II First Strand cDNA Synthesis Kit (New England, E6560S) following default protocol. The Envelope CDS was then amplified using custom primers in Q5 High-Fidelity DNA Polymerase PCR (New England Biolabs, M0491S) with 25 cycles, melting temperature of 53 C. In addition to the cellular cDNA, we performed PCR and library prep on the input library from each LongAmp M13 PCR product used in a virus production experiment. PCR products were purified using NEB kit and input into the Library Preparation 591 EF 2.0 with Enzymatic Fragmentation and Twist Universal Adapter System kit (TWIST Bioscience) targeting 300 bp libraries. Sequencing was performed using a NextSeq 1000/2000 P1 Reagent Kit, 300 cycle (Illumina, 20050264. Library prep and sequencing was done twice where each run multiplexed one replicate for each cell line as well as the input library for that round of virus production.