NextSeq 500 sequencing; RNAseq analysis of Arabidopsis Col-0 wild-type and fbl17 mutant seedlings
RNAseq analysis of Arabidopsis Col-0 wild-type and fbl17 mutant seedlings
Whole 10 day-old in vitro-grown seedlings are snap-frozen in liquid nitrogen before RNA extraction. Arabidopsis sterile seeds are plated on MES-buffered Murashige and Skoog) medium (1X MS salts; Duchefa; 0.8% agar, Fluka; 1% (w/v) sucrose; pH adjusted to 5.7 with KOH). Plated seeds are stratified at 4°C for 2-3 days and then transferred to a plant growth chamber under a 16-h-light/8-h-dark cycle (22/20°C) until collection.  Total RNAs were extracted from 10 day-old whole seedlings in vitro-grown by using Trizol solution (Invitrogen) completed by a second phenol/chloroform treatment. Three biological replicates were used as starting material. RNAs concentrations were determined with a QuBit Fluorometer (Thermo Fisher Scientific). RNAs integrities were checked using the 2100 Bioanalyzer (Agilent).  mRNAs were isolated from total RNAs by using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB) for mRNA libraries preparation. Sequencing libraries were prepared using the Collibri stranded RNA library kit for Illumina (Invitrogen).