Total RNA was extracted from plant tissues using a Directzol RNA kit (Zymo Research). Reverse transcription was performed with 1 μg total RNA with the qScript cDNA SuperMix (Quanta Biosciences). mRNA levels were assessed by quantitative PCR with primers described previously to detect telomere-related transcripts (Cifuentes-Rojas et al. 2011 (link); Leehy et al. 2013 (link)) or DDR transcripts (Boltz et al. 2012 (link); Cifuentes-Rojas et al. 2012 (link); Hashimura and Ueguchi 2011 (link)), using SsoAdvanced Universal Supermix (Bio-Rad). RNA from at least three individual plants was used for each genetic background and at least two technical replicates were run for each reaction. Expression levels were averaged and normalized to GAPDH. Wild-type was set to 1 and mutant samples were compared to this value. To detect transcription of retrotransposons, primers recognizing AtMu1 and ATGP3 were used as described previously (Hirochika et al. 2000 (link); Tsukahara et al. 2009 (link)).
TERRA was monitored by northern blot using 10 μg of total RNA. RNA was resolved on a 1% agarose gel, transferred to a nylon membrane and hybridized with a 32P 5′ labeled (CCC TAA A)5 probe as previously described (Zellinger et al. 2007 (link)).
TERRA was monitored by northern blot using 10 μg of total RNA. RNA was resolved on a 1% agarose gel, transferred to a nylon membrane and hybridized with a 32P 5′ labeled (CCC TAA A)5 probe as previously described (Zellinger et al. 2007 (link)).
