Polyattract system

Mouse embryonic fibroblasts (MEFs) of wild-type and FTO-4 C57BL/6J mouse embryos were cultured at 37°C and 7% CO₂ in Dulbecco's Modified Eagles Medium Glutamax (Gibco, Paisley, Scotland) supplemented with 10% (v/v) fetal calf serum, 100 units/ml penicillin, 100 units/ml streptomycin, 1x non-essential amino acids (Gibco, Paisley, Scotland) and 50 µM beta-mercaptoethanol (Gibco, Paisley, Scotland). Total RNA was isolated using an RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's instructions. An aliquot of total RNA was taken for subsequent analysis. The remainder of the total RNA was used for mRNA extraction with the polyATtract system (Promega).

P. syringae 61 hrcC-treated or non-treated control leaves were ground under liquid nitrogen 3, 6 and 48 hours after inoculation. Equal amounts of 3- and 6-hour samples were pooled. Total RNA was extracted using the Plant Total RNA Extraction Miniprep System (Viogene). mRNA was obtained from 100 µg total RNA for driver and tester each, using the PolyATtract System (Promega) as recommended by the manufacturer.
cDNA production and subtractive hybridization were performed using PCR Select cDNA Subtraction Kit (Clontech) as recommended by the manufacturer. Uninoculated plant material served as “driver” and inoculated plant material as “tester”. Cloning of subtracted fragments was carried out using the TOPO TA Cloning Kit for Sequencing (Invitrogen).