title: Illumina HiSeq 1500 paired end sequencing; Strategies for in vivo screening and mitigation of toxicities associated with antisense therapeutics
design: Strategies for in vivo screening and mitigation of toxicities associated with antisense therapeutics
library_construction_protocol: Acute dose screens (3 day). All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals. Male Crl:CD1(ICR) mice were obtained from Charles River UK Ltd, Margate, Kent. For both acute studies, adult, \"wild type\" male CD1 mice were intravenously dosed with 100 mg/kg of each of the ASOs on day 1. The exception was ‘Sequence 1’, which was only administrated at 30 mg/kg due to concerns over its tolerability at a higher dose. A dose of 100 mg/kg was selected as it was below the total cumulative dose that was administrated in the 15 day repeat dose study, but still anticipated to be sufficient for the more hepatotoxic ASOs to cause ALT and AST increases. Four to five mice per group were dosed on day 1 (5 mL/kg), with ASOs formulated in 1x PBS (pH 7.4) at a 20 mg/mL and 6 mg/mL, for 100 mg/kg and 30 mg/kg doses respectively.  Total RNA was extracted from ~50 mg of snap-frozen liver using RNeasy Plus Mini Kit (Qiagen)(starting with 800 µL of Buffer RLT Plus and extracting the RNA from 350 µL of lysate) and MagNA Lyser Green Beads (Roche)(tissues were shredded for 33 sec at 6200 rpm). Samples were then quantified and their integrity assessed using Qubit RNA Broad Range Assay Kit (Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer RNA 6000 Nano Kit (Agilent Technologies) respectively. The extracted RNA had an average RNA integrity number (RIN) score of 8.9 and no sample with a RIN score < 8.1 was used for sequencing.  RNA-Seq libraries were prepared with a TruSeq® Stranded Total RNA LT - (with Ribo-Zero™ Human/Mouse/Rat) - Set A kit (Illumina). The library preparation followed the manufacturer’s manual except for two modifications made to the thermal cycler parameters. First, the time of ‘Elution 2 – Frag – Prime’ program was reduced from 8 to 6 min to increase the length of the RNA fragments. Secondly, the number of cycles (11 instead of 15) and the time of the initial step in each cycle (30 instead of 10 sec at the first 98°C step) were both modified to minimise PCR-based amplification bias (‘PCR’ program). Three rounds of qPCR quantification of the final libraries were performed with the KAPA Library Quantification Kit (KAPA Biosystems) on a QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific).
attributes.tag: Experimental Factor: M30472N
attributes.value: study
attributes.tag: Experimental Factor: acute dosing
attributes.value: protocol
attributes.tag: Experimental Factor: GSK2910584A
attributes.value: compound
attributes.tag: Experimental Factor: 100
attributes.value: dose
attributes.tag: Experimental Factor: 3
attributes.value: time