BioSample

Protocols: Cell lysis and RNA isolation were performed using the Total RNA Purification Kit (Norgen Biotek Corporation 17200) according to the manufacturer's protocol. On-column DNA removal was conducted using Norgen's RNase-Free DNase I Kit (Norgen Biotek Corporation 25710) following the manufacturer's instructions. Cell lysis and RNA isolation were performed using the Total RNA Purification Kit (Norgen Biotek Corporation 17200) according to the manufacturer's protocol. On-column DNA removal was conducted using Norgen's RNase-Free DNase I Kit (Norgen Biotek Corporation 25710) following the manufacturer's instructions. The initial RNA was QCed using Agilent Bioanalyzer with the RNA Nano Assay kit as per the manufacturer’s protocol. The RNA sample set was then standardized to 300 ng total RNA in 50 µl using the concentration values given by the Bioanalyzer. The libraries were prepared on a Beckman Coulter Automated Workstation Biomek i7 Hybrid (MC +Span-8). For library preparation an automated version of the NEBNext® UltraTM II Directional RNA Library Prep Kit was used, following section 1 - Protocol for use with NEBNext Poly(A) mRNA Magnetic Isolation Module. An adaptor dilution of 1 to 20 was used, the samples were individually barcoded using unique dual indices during the PCR using 13 PCR cycles as per the manufacturer’s protocol. The individual libraries were quantified using the Qubit HS DNA assay as per the manufacturer’s protocol. For the measurement 1ul of sample in 199ul of Qubit working solution was used. The quality and molarity of the libraries was assessed using Agilent Bioanalyzer with the DNA HS Assay kit as per the manufacturer’s protocol. The assessed molarity was used to equimolarly combine the individual libraries into one pool for sequencing.