GSM5602309: HB37: 14 dpi SARS-CoV-2; Macaca mulatta; RNA-Seq
collection protocol: Whole venous blood was collected from nonhuman primates using a BD Vacutainer Safety-Lok Blood Collection Set (BD catg#367281) and BD Vacutainer one use needle holder (BD catg# 364815). Blood was added to the PAXgene Blood RNA reagent tube (PreAnalytiX catg#762165) in the same ratio as the manufacturer's guidelines. Post collection, PAXgene blood tubes were stored upright for a minimum of two hours and then placed into the -20C freezer until further processing. Samples were processed in accordance with the PreAnalytiX PAXgene Blood RNA Kit (PreAnalytiX catg#762165). The RNA Clean & Concentrator-5 kit (Zymo, Catg#R1015) was used to purify and increase RNA samples with low yields. Procedures were followed per manufacturer's guidelines. For AGM NC33, Paxgene tubes were not collected at 1 and 3 dpi due to blood being prioritized for other assays. Total RNA samples were quantitated using Qubit RNA HS Assay kit (Invitrogen Q32855). A RIN for each sample was determined by running 1ul on an Agilent 4150 TapeStation using RNA Screen Tapes (Agilent 5067-5576). 1-2 ug of each sample were treated with Baseline Zero DNase (Epicentre, DB0711K) for 30 minutes at 37°C. DNase was inactivated by adding 2ul 10X Stop solution and following a 10 minute incubation at 65°C. The Total RNAs were then purified using Agencourt RNAClean XP magnetic beads by following the manufactory recommendation (Beckman Coulter Life Sciences). 10 ng of each sample were used to make SMART-Seq libraries following the SMART-Seq Stranded Kit User Manual (Takara Bio). Briefly, cDNAs were first generated from all total RNA fragments after RNA fragmentation. Addition of Illumina adapters and indexes and then library purification were followed. Final library amplification and purification were performed after enzymatically removing ribosomal fragments originating from rRNA molecules by using probes specific to mammalian rRNA. Libraries were quantitated using Qubit DNA HS Assay kit (Molecular Probes Life Technologies, Q32854). Average size of each library was determined by running each sample on Agilent 4150 TapeStation using D1000 Screen Tapes. (Agilent 5067-5582). Each library was diluted to 4nM in DNase RNase free ultrapure water (Invitrogen 10977-015). All libraries were pooled at a concentration of 4nM before denaturing with 0.2 N NaOH for 5 min at RT. The libraries were neutralized using 0.2 N Tris-HCl, pH 7 before being diluted to 20pM in HT1 buffer (Illumina). Finally, denatured libraries were loaded into an Illumina NextSeq 550 v2 High-Output reagent cartridge at a final concentration of 1.2pM in HT1 buffer. Denatured PhiX control library was also included at 1% concentration. Dual indexes and paired end 75bp sequencing was performed on a High-Output flow cell yielding about 18M read pairs per sample.