RNA-Seq of RVFV
Total nucleic acid was depleted of host genomic DNA by DNase treatment using the TURBO DNase kit (Invitrogen, CA, USA) by incubating at 37 C for 30 minutes. The RNA was cleaned using RNAClean XP (Beckman Coulter, USA) and used to synthesize the first complementary DNA (cDNA) strand using SISPA random primers proposed by Djikeng19 (FR26.RV 5`GCCGGAGCTCTGCAGATATCNNNNNN 3`) with Superscript IV first strand synthesis kit (Invitrogen, CA, USA). The second strand cDNA was synthesized by a Klenow reaction (New England Biolabs, MA, USA). Random fragment amplification was done using MyTaq Red Mix master mix (Meridian Bioscience Inc, OH, USA) with 10 mM of primer FRFR20.RV (5`GCCGGAGCTCTGCAGATATC 3`) with the following thermal regimen: 1 cycle of 98 C for 30 sec, followed by 38 cycles each of 98 C for 10 sec, 54 C for 20 sec and 72 C for 45 sec. A final extension step of 72 C for 5 min was included, followed by a 4 C incubation. The random amplicons were then cleaned with Agencourt Ampure XP beads (Beckman, CA, USA) and used to prepare sequence libraries using Collibri ES DNA library prep kit (Invitrogen, CA, USA). The generated DNA libraries were quantified using a Qubit dsDNA HS Assay kit (Invitrogen, CA, USA) and the library size measured on the 4200 TapeStation System (Agilent Technologies, CA, USA). The pooled library was adjusted to 4 nM concentration and then denatured with 0.2 N NaOH and further diluted to 12 pM. 10% PhiX control library (Illumina, CA, USA) was spiked into the pool, and the library sequenced in pairs using the 600 v3 paired end chemistry on a Miseq (Illumina, CA, USA).