title: GSM9429002: enriched, WNV, 6dpi, 2; Mus musculus; OTHER
library_construction_protocol: Samples were homogenized using homogenization buffer (50 mM Tris, pH 8.0; 100 mM KCl; 12 mM MgCl2; 1 % Igepal; 1 mg/mL sodium heparin; 100 µg/mL cycloheximide; 0.2 U/µL SUPERaseIn RNase Inhibitor, 2 mM DTT). After centrifugation (10,000 x g; 10 min; 4oC) 25 µg of primary antibody against GFP (rabbit; # AB290; Abcam) was added, and samples were rotated for 1 h at 4 oC. Subsequently, 200 µl of DynabeadsTM Protein G (# 10004D; Invitrogen) were added, and samples were incubated rotating overnight at 4 C. The beads were washed four times with high-salt buffer (50 mM Tris, pH 8.0; 300 mM KCl; 12 mM MgCl2; 1 % Igepal; 100 µg/mL cycloheximide). RNA was extracted and purified with Arcturus PicoPure RNA Isolation Kit (ThermoFisher: Kit0204 - Applied biosystems; 12204-01) according to the manufacturer's instructions. To prevent contamination with DNA, RNase-Free DNase Set (Qiagen; # 29254) was used according to the manufacturer's instructions. Library preparation was performed with 1-50 ng of total RNA.  cDNA was prepared using the SeqPlex RNA Amplification Kit (Sigma) per manufacturer's protocol and then fragmented using a Covaris E220 sonicator using peak incident power 18, duty factor 20%, cycles per burst 50 for 120 seconds. cDNA was blunt ended, an A base was added to the 3' ends, and then Illumina sequencing adapters were ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.