GSM8141447: prt-16hyt1-1, 7-d-old continuous light grown seedlings, Biological replicate 4; Arabidopsis thaliana; RNA-Seq
Total RNA was extracted using RNAeasy minikit and sent for RNA sequencing RNA was extracted using RNeasy plant mini kit Qiagen as per manufacturer's instructions. RNA was sent to Deep-Seq Next Generation Sequencing Facility (University of Nottingham) for further analysis. RNA concentrations were measured using the Qubit Fluorometer and the Qubit RNA BR Assay Kit (ThermoFisher Scientific; Q10211) and RNA integrity was assessed using the Agilent 4200 TapeStation and the Agilent RNA Screentape (Agilent; 5067-5576). Poly(A) selection was performed on 1 μg of total RNA, using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB; E7490L). Indexed sequencing libraries were then prepared using the NEBNext Ultra Directional RNA Library Preparation Kit for Illumina (NEB; E7420) and NEBNext Multiplex Oligos for Illumina, Index Primer Sets 2, 3 and 4 (NEBNext; E7500S, E7710S and E7730S). PolyA selection, cDNA synthesis and library generation were carried out using the Biomek 4000 Automated Laboratory Workstation (Beckman Coulter). Libraries were quantified using the Qubit Fluorometer and the Qubit dsDNA HS Kit (ThermoFisher Scientific; Q32854). Library fragment-length distributions were analysed using the Agilent TapeStation 4200 and the Agilent High Sensitivity D1000 ScreenTape Assay (Agilent; 5067-5584 and 5067-5585). Libraries were pooled in equimolar amounts and final library quantification performed using the KAPA Library Quantification Kit for Illumina (Roche; KK4824). The library pool was sequenced on the Illumina NextSeq500 using two NextSeq500 High Output 150 cycle kits