GSM5014165: L3; Chlorocebus sabaeus; Severe acute respiratory syndrome coronavirus 2; RNA-Seq
For crosslinking, cells or virus pellet were incubated with 2 mM of EZ-Link Psoralen-PEG3- Biotin (ThermoFisher Scientific) at 37℃ for 10 min in PBS containing 0.01% digitonin. The cells were then spread onto a 10cm plate and irradiated using 365 nm UV for 20 min on ice. Cell and virion RNA were extracted with RNeasy mini kit (Qiagen). RNA was fragmented using RNase III (Ambion) and purified using 2×MagicPure RNA Beads (TransGen). Each RNA sample was divided in two: one half was used for proximity ligation and then crosslink reversal (C, L and V samples), while in the other half, crosslink reversal was done before proximity ligation (non-ligated C_N, L_N, V_N). Proximity ligation was done under the following conditions: 200ng fragmented RNA, 1 unit/μl RNA ligase 1 (New England Biolabs), 1x RNA ligase buffer, 50 mM ATP, 1 unit/μl Superase-in (Invitrogen), final volume: 200 μl. Reactions were incubated for 16 hours at 16 °C and were terminated by cleaning with miRNeay kit (Qiagen). Crosslink reversal was done by irradiating the RNA on ice 254 nm UVC for 5min using a CL-1000 crosslinker (UVP). Sequencing libraries were prepared with 50ng input RNA material using SMARTer Stranded Total RNA-seq Kit v2—Pico Input Mammalian (Takara Bio USA Inc., USA), according to the manufacturer's instructions. The libraries were paired-end sequenced (PE150) using Illumina Nova seq platform.