title: NextSeq 500 sequencing; Analysis of Zika Virus gene expression by Ribosome profiling and RNA sequencing
design: Analysis of Zika Virus gene expression by Ribosome profiling and RNA sequencing
library_construction_protocol: Vero E6 cells and C6/36 cells were maintained in Dulbecco's modification of Eagle's medium supplemented with 10% (vol/vol) fetal calf serum (FCS). ZIKV strain Pe243 (ZIKV-PE243) was derived as previously described (Donald et al., 2016). At 24 h p.i., cells were treated with CHX (Sigma-Aldrich; to 100 µg/ml; 2 min). Cells were rinsed with 5 ml of ice-cold PBS, the dishes submerged in a reservoir of liquid nitrogen for 10 s, transferred to dry ice and 400 µl of lysis buffer [20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 µg/ml cycloheximide and 25 U/ml TURBO™ DNase (Life Technologies)] dripped on. Cells were scraped extensively to ensure lysis, collected and triturated with a 26-G needle ten times. Lysates were clarified by centrifugation for 20 min at 13,000 g at 4 ºC, the supernatants recovered and stored in liquid nitrogen. Cell lysates were subjected to RiboSeq and RNASeq. The methodologies employed were based on the original protocols of Ingolia and colleagues (Ingolia et al., 2009; Ingolia et al., 2012), except ribosomal RNA contamination was removed by treatment with duplex-specific nuclease (DSN) for Samples 7 and 8, and library amplicons were constructed using a small RNA cloning strategy (Guo et al., 2010) adapted to Illumina small RNA v2 to allow multiplexing. The methods used were as described by Irigoyen et al. (2016).
attributes.tag: Experimental Factor: Vero E6
attributes.value: cell line