Ambion rnaqueous micro kit

Drops of worms fixed in 70% methanol were pipetted onto a Leica PEN membrane slide (76 × 26 mm P/N 11505158). The slide was then placed on a slide warmer at 50°C to evaporate the methanol. Once dried, the slide was mounted onto a Leica laser dissection microscope. Costar Thermowell tubes (0.5 ml with flat cap, Cat#: 6530) were loaded into the collection holder, and 65μL of lysis buffer (Ambion RNAqueous-Micro Kit, ThermoFisher Scientific, cat # AM1931) was pipetted into the tube cap. Using the 40x objective, 50–100 tail tips were dissected into each cap. Before unloading the tube from the collection holder, an additional 35μL of lysis buffer was added to the tube cap. Tubes were spun down, flash frozen in liquid nitrogen or on dry ice and stored at −80 °C until enough tissue was collected for one RNA extraction (240–440 tail tips). RNA was extracted using the Ambion RNAqueous-Micro Kit following the manufacturer’s protocol and eluted twice into 8 μl elution solution. The quality and quantity of the extracted RNA was evaluated with Agilent high sensitivity RNA screen tapes (#5067–5579).

RNA was isolated using the Ambion^® RNaqueous^® micro kit (ThermoFisher Scientific) according to manufacturer’s instructions. RNA quality was analyzed using the Agilent 6000 Pico LabChip^® and Agilent 2100 Bioanalyzer software (Agilent Technologies, USA). RNA quantity was determined using the Quant-iT™ RiboGreen^® kit (ThermoFisher Scientific) according to manufacturer’s instructions. NEBNext Oligo d(T)₂₅ beads (New England BioLabs, Ipswich, MA, USA) were used for mRNA purification. cDNA library synthesis was performed according to Ziegler et al.^{64 (link)} with three biological replicates per treatment each containing three A. thaliana leaves. Subsequently, 100 bp paired-end RNA sequencing was performed on the Illumina HiSeq4000 platform (Génome Québec Innovation Centre, McGill University, Montreal, Canada).