4SU-Seq of KSHV lytic reactivation in iSLK-BAC16 model

Subconfluent monolayers of iSLK-BAC16 (WT or deltaORF57) were induced with 1 ug/mL Doxycycline, 1 mM Sodium Butyrate in 1x DMEM media supplemented with 2% Tet-approved FBS. 0 hour time point was when induction media was added and cells were first placed at 37 degrees Celsius to incubate. At indicated times relative to induction, 10 mM 4SU was added to cell culture medium. 15 minutes post-4SU addition, cells were collected and RNA extracted. 40-50 ug total RNA was biotinylated in 10 mM Tris pH 7.4, 1 mM EDTA, 0.2 mg/mL EZ-link Biotin-HPDP. Unbound biotin was removed by performing a chloroform:isoamyl alcohol extraction and isopropanol precipitation. Biotinylated RNA was bound to My One Streptavidin T1 Dynabeads. Bound beads were washed three times and then 4SU-RNA was eluted with 100 mM DTT. Pulldown RNA was ribominus selected using the NEBNext rRNA Depletion Kit v2 and RNA-Seq libraries were generated using the NEBNext Ultra II Directional RNA Library Prep Kit. Two biological replicates were sequenced using the Illumina NextSeq 550 platform to generate 150 bp PE reads.