RNA-sequencing of transgenic Arabidopsis seedlings constitutively expressing a Colletotrichum higginsianum effector
RNA-sequencing of transgenic Arabidopsis seedlings constitutively expressing a Colletotrichum higginsianum effector
Seven 19-day-old whole seedlings (equating to 70-90 mg of fresh weight per sample) were removed from sterile plates and immediately frozen in liquid nitrogen. Seedlings were grown vertically on GM agar plates [4.4 g/L Murashige Skoog Medium (Duchefa M0222), 0.50 g/L MES hydrate pH6 (Sigma M8250), 20 g/L agar (Difco 214030)] under long-day conditions (16 h light/8 h dark photoperiod) at 20 °C. Frozen seedlings were homogenised using a 2010 Geno/Grinder (SPEX SamplePrep) at 1200 rpm for 1 min. RNA was extracted using the RNeasy Plant Mini Kit (QIAGEN) using buffer RLT according to the manufacturer's instructions with the following modification: after tissue homogenisation using the QIAshredder column, two or three centrifugation steps were performed to maximise removal of debris. RNA was eluted from RNeasy spin columns in 30 µl of H2O. RNA extracts were DNase-treated using the TURBO DNA-free^TM Kit (Invitrogen) according to the manufacturer's rigorous procedure. Total RNA samples were submitted to Genewiz for library preparation with polyA selection and standard RNA-sequencing. 