Fig. 4

SVALKA transcription mediates transcription activity antisense to CBF1. a Representative Northern blot of a cold exposure time series of Col-0 (WT) and hen2-2. The probe used for asCBF1 is shown in b. Blots were repeated with three biological replicates with similar results. UBI is used as loading control. Uncropped blots can be found in the Source Data file. b Graphical representation of the CBF1-SVK genomic region. The probe for asCBF1 is shown in red. RT was done with an oligo-linked primer to ensure strand-specificity and generated cDNA was used in c. c RT-qPCR of asCBF1 after cold exposure in hen2-2 and the double mutants, hen2-2uns-1, and hen2-2svk-1. The RT-primer and the qPCR primers are shown in the graphical representation above the graph. Bars represent mean (white: 4 h 4 °C, black: 8 h 4 °C, ±SEM) from three biological replicates (rings). The relative level of asCBF1 was normalized to the level in hen2-2 after 4 h of 4 °C. Statistically significant differences were determined with Student’s t-test (***p < 0.001). Source data are provided as a Source Data file. d) RT-qPCR of SVK read-through transcripts. Hundred nanograms of RNA from an RNAPII-IP (i.e. RNA attached to RNAPII) or total RNA was converted to cDNA with an oligo-linked primer. The RT-primer and qPCR primers used can be seen in the graphical representation above the graph. The RT-primer annealed to a sequence downstream of the PolyA signal of SVK. A read-through transcript can be detected in the nascent RNA samples with an increased expression after 8 h of cold exposure. No signal could be seen in the Mock-IP or total RNA sample. Each bar represents the mean (white: 4 h 4 °C, black: 8 h 4 °C, ±SEM) from three biological replicates (rings). The relative level of read-through transcripts was normalized to the level in the nascent sample after 4 h of 4 °C. Statistically significant differences were determined with Student’s t-test (***p < 0.001). Source data are provided as a Source Data file