RIP-SEQ of DZIP3 in GFP-VSV infected HEK293T cells
RIP-SEQ of DZIP3 in GFP-VSV infected HEK293T cells
Flag-tagged plasmids transfected in HEK293T cells were infected with GFP-VSV (MOI: 1) for 6 hours. Cells were washed with 1X ice-cold PBS and resuspended in equal pellet volumes of polysome lysis buffer (100 mM KCl, 5mM MgCl2, 10 mM HEPES-NaOH(pH-7.0), 0.5% NP-40, 0.1mM DTT, 20 Units/ml RNase Inhibitor, and EDTA-free Protease Inhibitor cocktail). The lysates were then incubated on ice for 30 mins and stored at -80 °C overnight. Protein G Dynabeads were incubated with 5 µg of Flag-antibody in NT-2 buffer (50mM Tris-HCl, pH-7.4, 150mM NaCl, 1mM MgCl2, 0.05% NP-40) at 4 °C for 12 hours. Beads were washed with NT-2 buffer and resuspended in NET-2 buffer (1X NT-2 buffer, 20mM EDTA, pH-8.0, 1mM DTT, 20 units/ml RNase inhibitor). Cell lysates were centrifuged at 20000 xg for 20 mins at 4°C and incubated with antibody-bound beads at 4°C. After 12 hours, cells were washed with NT-2 buffer and resuspended in Proteinase K buffer (1X NT-2 buffer with 1% SDS, 1.2 mg/ml Proteinase K). The cell lysates were then incubated at 55 °C for 30 mins, and proceeded for RNA isolation using phenol:chloroform:isoamyl alcohol (25:24:1) method. RNA was precipitated in 0.25 M ammonium acetate, 0.12 M LiCl, 25 mg of glycogen, and absolute ethanol (Final volume 1 ml) at -80 °C for overnight. Next day, RNA was collected and treated with DnaseI for one hour at RT, followed by library preparation and sequencing. The Library was prepared using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® kit (E7760L).