CLIP-seq for ZAP in HEK293T control and ZAP-L or ZAP-S isoform knockout cells infected with wild type HIV-1 or CpG-recoded HIV-1
CLIP-seq for ZAP in HEK293T control and ZAP-L or ZAP-S isoform knockout cells infected with wild type HIV-1 or CpG-recoded HIV-1
Cells were infected at MOI=3 and media was replaced 18 h post-inoculation with DMEM supplemented with 2% fetal bovine serum. At 48hpi, cells were washed with ice-cold PBS, irradiated once with 150 mJ/cm2 in a Stratalinker 2400 at 254 nm, harvested by scrapping in PBS, pelleted by centrifugation then resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4; 100 mM NaCl; 1% Igepal CA-630; 0.1% SDS; 0.5% sodium deoxycholate; protease inhibitor). The nitrocellulose membrane strips were digested with 10μl proteinase K in 200μl proteinase K SDS buffer (10mM Tris-HCl pH 7.4, 100mM NaCl, 1mM EDTA, 0.2% SDS) for 1 hour at 50oC. The supernatant was transferred to a phase lock gel heavy tube. The RNA extracted were then phenol/chloroform extracted from the supernatant by adding 200μl of Phenol:Chloroform:Isoamyl Alcohol 25:24:1, incubating shaking for 5 min at 30°C, and centrifuged to separate the aqueous and organic phases. The aqueous phase was further extracted with 0.8ml of chloroform, centrifuged, and the upper phase containing the RNA is transferred to a new tube, and then precipitated with 20μl 3M NaAc, 0.75μl glycoblue and 0.5ml EtOH overnight at −20°C. The RNA is pelleted for 30 minutes at 4°C at 13000G. Custom individual cross-linking and immunoprecipitation (iCLIP) library construction protocol.