E-MTAB-6180:EV_6hpi.rep2_s
NextSeq 550 sequencing; Ribo-Seq transcriptomic analysis of human RD cells infected with enterovirus strains EV7, EV71, and PV1.
Ribo-Seq transcriptomic analysis of human RD cells infected with enterovirus strains EV7, EV71, and PV1.
EV_6hpi.rep2_s
RD cells were grown on 150-mm dishes to reach 90% confluency and infected at MOI 20 with EV7, PV1 or EV71 virus stocks. At indicated times postinfection, cells were treated with 3 mM cycloheximide for 3 min, flash frozen in a dry ice/ethanol bath, and lysed in the presence of 100 µg/ml cycloheximide. Cell lysates were subjected to Ribo-Seq based on the previously described protocols (Irigoyen et al. 2016; Ingolia et al. 2012). Cells were rinsed with 10 ml of ice-cold PBS, the dishes were freezed for 45 s in dry ice / ethanol bath, transferred to ice and 600 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 27-G needle ten times. Lysates were clarified by centrifugation for 20 min at 13,000 g at 4 ºC, the supernatants recovered and stored at -80 ºC.  Cell lysates were subjected to RiboSeq. The methodologies employed were based on the protocols described before. Briefly, cells are lysed under conditions optimised to minimise further ribosome movement (addition of translation inhibitors, rapid freezing), the lysate is treated with ribonuclease (often RNase 1) to degrade regions of mRNAs that are not physically protected, and the ribosomes harvested on sucrose gradients or through a sucrose cushion. The ribosome pellet is de-proteinized, the ribosome-protected fragments (RPFs) harvested by elution from a polyacrylamide gel, ligated to adapters, subjected to RT-PCR, deep sequenced and mapped back to the genome. This analysis reveals the location and abundance of ribosomes on mRNAs with up to single-nucleotide precision. The corresponding transcriptome is also determined from the same lysate: total RNA is harvested, fragmented, cloned and sequenced to generate a parallel RNA sequencing (RNASeq) library. (Irigoyen et al., 2016 doi: 10.1371/journal.ppat.1005473, Ingolia et al., 2012 doi:  10.1038/nprot.2012.086).
University of Cambridge
E-MTAB-6180:EV_6hpi.rep2_s
Experimental Factor: sampling time point
6
Experimental Factor: infect
Echovirus 7