Identification of RNase L-Dependent, 3'-End-Modified, Viral Small RNAs in Sindbis Virus-Infected Mammalian Cells

Girardi, Erika; Chane-Woon-Ming, Béatrice; Messmer, Mélanie; Kaukinen, Pasi; Pfeffer, Sébastien

Small RNA cloning and sequencing. RNA was extracted from noninfected and SINV-infected HEK293 and Vero cells 16 hpi. Small RNA cloning was conducted with 10 or 20 g of total RNA as previously described (14, 29) . Small RNA libraries from SINV-infected HEK293 cells treated with control or RNase L-specific siRNAs were generated following the same protocol with some modifications. Mainly, small RNAs were ligated using degenerate 5= and 3= adapters to limit biases in the ligation step (56) . This procedure was also used to prepare small RNA libraries after NaIO 4 treatment of 100 g of total RNA, as previously described (57) , except that the 3= ligation was performed at 16°C overnight, in 12% polyethylene glycol 8000 (PEG 8000) to favor the cloning of modified small RNAs. Sequencing was performed at the IGBMC Microarray and Sequencing platform, Illkirch, France, using different Illumina instruments (Genome Analyzer IIx, HiSeq 2000, and HiSeq 2500) with a read length of 36 or 50 bp.