GSM4850316: ZIKV_AGO_CLIP_Rep2; Homo sapiens; OTHER
For AGO-CLIP, AGO HITS-CLIP was previously utilized to map AGO-bound miRNA (Chi et al., 2009; Clark et al., 2014). To discriminate functional ZIKV-derived viRNAs from ZIKV genome, we conducted the crosslinking and immunoprecipitation (CLIP) with minor modifications. Briefly, NSCs were grown to 90% confluency and infected ZIKV at a MOI of 1 for 48 h. At 24 hpi, Proten A Dynabeads (Invitrogen) were washed 3 times with PBS and resuspended in antibody binding (AB) buffer (1×PBS, 0.02% Tween-20). Per cell sample, 150 μl of the beads was used. Beads were rotated with 10 μl of bridging antibody (rabbit anti-mouse IgG, Jackson Immunoresearch) for 30 min at room temperature. Beads were then washed 3 times in AB buffer before adding 8 μl of pan-Ago antibody (2A8, EMD Millipore). Beads were then rotated for overnight at 4ºC. After 48 h post ZIKV infection, NSCs were washed twice with PBS and UV irradiated at 254 nm for a total energy dispersion of 600 mJ/cm2 in a Stratlinker XL-500 (Stratagene). Cells were lysed with 1×PXL (1×PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% NP-40, protease inhibitors). Cell lysates were treated sequentially with RNAsin (Promega) and RQ1 DNase (Promega) prior to co-immunoprecipitation of RNA-protein complexes on the protein A Dynabeads for 4 h at 4ºC. Beads were then washed twice each with 1×PXL, 5×PXL (5×PBS, 0.1% SDS, 0.5% deoxycholate, 0.5% NP-40, protease inhibitors), and 1×PNK buffer (50mM Tris-Cl, 10mM MgCl2, 0.5% NP-40, protease inhibitors). CLIP-RNAs were liberated with their on-bead protein complexes by treatment with 4 mg/ml protease K (Sigma) and subsequent phenol/chloroform extraction as described previously (Chi et al., 2009). miRNA libraries of CLIP-RNA were constructed and sequenced on HiSeq3000.