Quantitative Analysis of FLC Antisense Transcripts in Arabidopsis

RNA was extracted from seedlings using Spectrum™ Plant Total RNA Kit (Sigma) and treated with TURBO DNA-free Kit (Ambion) to remove any genomic DNA contaminants. Purified RNA (500 ng) is subjected to cDNA synthesis using RevertAid Reverse Transcriptase (Thermo) with Oligo(dT)₁₈ primer. The synthesized cDNA was used for PCR and qPCR analyses. PCR products were analyzed by gel electrophoresis and the PCR band intensities were quantified using ImageJ. The qPCR analyses were performed with the SensiMix™ SYBR® & Fluorescein Kit (Bioline) on a LightCycler 480 (Roches). For each sample, two technical replicates were performed. The comparative cycle threshold method was used for calculating transcript level. Primers used for FLC antisense analysis are the same as in the previous publication^{37 (link)}. Sequences of oligo primers are listed in Supplementary Data 4.