GSM7743906: Dicer WT, SINV-GFP MOI 2 12 hpi, rep2; Homo sapiens; Sindbis virus; RNA-Seq
Cells were collected in Trizol reagent (Invitrogen, Thermo Fisher Scientific) and RNAs were isolated according to the manufacturer's instructions. ssRNA-seq (stranded RNA-seq): RNA-Seq libraries were generated according to the manufacturer's instructions from 250 ng of total RNA using the Illumina Stranded mRNA Prep, Ligation kit (Reference Guide - PN 1000000124518) and IDT for Illumina RNA UD Indexes Ligation (Illumina, San Diego, USA). Briefly, Oligo(dT) magnetic beads were used to purify and capture the mRNA molecules containing polyA tails.The purified mRNA were then fragmented at 94°C for 2 min and copied into first strand complementary DNA (cDNA) using reverse transcriptase and random primers. Second strand cDNA synthesis further generated blunt-ended double-stranded cDNA and incorporated dUTP in place of dTTP to achieve strand specificity by quenching the second strand during amplification. Following A-tailing of DNA fragments and ligation of pre-index anchors, PCR amplification was used to add indexes and primer sequences and to enrich DNA libraries (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 12 cycles; 5 min at 72°C). Surplus PCR primers were further removed by purification using SPRIselect beads (Beckman-Coulter, Villepinte, France) and the final libraries were checked for quality and quantified using capillary electrophoresis.