GSM3900212: Ribo_siP_2; Homo sapiens; OTHER
A549 cells were plated at 1.5 x 106 cells per 10 cm dish. Three 10cm dishes were transfected with NSC siRNA whereas three other dishes were transfected with either siP1_1, siP2_1 or siP2_4 siRNAs as described in the transfections section. 48 h later cells were infected as described previously with DENV-2 (NGC strain) at MOI of 10 in a total volume of 10 ml, rocked every 15 minutes for 1 h and the infection was allowed to proceed for more 1.5 h (2.5 h total time). Cells were then flash frozen in liquid nitrogen without cycloheximide pretreatment and cold lysis buffer containing CHX was used to lyse the cells on ice. The RIBOseq strategy was adapted from Ingolia and colleagues with a few modifications described next. After nuclease digestion, samples were run in a polysome gradient and the ribosome fractions were collected. As described by Reid and colleagues, fractions were extracted using Trizol LS (Thermo Fisher Scientific), and rRNAs were removed using the Ribo-Zero gold rRNA removal kit (Illumina, San Diego CA) according to the manufacturer's protocol. For adapter ligation and library building we used NEBNext Small RNA Library Prep Set (Illumina).
GEO Accession
GSM3900212