Ampure rna clean beads

Patient samples were taken by clinical staff using appropriate personal protective equipment. Inactivation of samples occurred in their country of origin. Samples were then shipped to the Broad Institute or tested at the local center (Nigeria, Sierra Leone). Samples were inactivated in AVL buffer (Qiagen) or TRIzol (Life Technologies) following standard operating procedures. Samples were stored in liquid nitrogen or at −20 °C. RNA was isolated at the clinical site using the QIAamp Viral RNA Minikit (Qiagen) according to the manufacturer’s protocol. Poly(rA) and host rRNA were depleted using RNase H selective depletion, using 616 ng oligo (dT) (40 nt long) and/or 1000 ng DNA probes complementary to human rRNA. Samples then underwent RNase-free DNase using a kit (Qiagen) according to the manufacturer’s protocol. AMPure RNA clean beads (Beckman Coulter Genomics) were used to clean and concentrate samples. cDNA synthesis was performed using the Superscript III kit (Thermo Fischer) plus dNTPs, random primers, and SUPERASE-IN for first-strand synthesis. Then, the 10× second-strand buffer kit (New England Biolabs), plus Escherichia coli DNA ligase, E. coli DNA polymerase, Rnase H, and dNTPs were used for second-strand synthesis. Samples then underwent a final AMpure DNA beads clean-up^{30 (link)}.

RNA was extracted from 140 μl of CSF using the QIAamp Viral RNA Mini Kit (Qiagen) followed by Turbo DNAse treatment (Ambion) and purification with Agencourt RNAClean XP beads. We next depleted host rRNA using custom probes and RNAse H treatment as previously described^{25 (link)}. Depleted samples were purified using AMPure RNA clean beads (Beckman Coulter Genomics) and eluted in 10 ul of RNase-free water for cDNA synthesis. RNA was converted to double-stranded cDNA in two steps. First, RNA was retro transcribed using random primers and SuperScript IV (Invitrogen). Second-strand cDNA was generated using E. coli DNA ligase, RNAse H and DNA polymerase (New England Biolabs) and purified using Agencourt AMPure XP beads (Beckman Coulter). Libraries were then prepared using the Nextera XT DNA Library Prep Kit (Illumina) and sequenced on an Illumina NextSeq500 (2 × 75 cycles).