Illumina HiSeq 4000 sequencing; RNA seq of Arabidopsis Class II TPS mutants compared to wt, in ZT4, ED (end of day), EN (end of night) and ExtN (extended night)
RNA seq of Arabidopsis Class II TPS mutants compared to wt, in ZT4, ED (end of day), EN (end of night) and ExtN (extended night)
At day 28, whole rosettes were harvested in the light and immediately quenched in liquid nitrogen, with four or five plants from the same pot being pooled for each biological replicate. Frozen plant tissue was ground to a fine powder at -70°C in a robotized cryogenic grinder (Labman Automation Ltd, http://www.labman.co.uk) and stored at -80°C until use. Arabidopsis seeds were germinated on soil mixed with vermiculite (1:1) as described by Martins et al. (2013). After germination (7 days), seedlings were transplanted to 10-cm diameter pots (four or five seedlings per pot), and grown in a controlled environment chamber (Percival Scientific, http://www.percival-scientific.com) at a constant temperature of 20°C for 21 more days. Total RNA was extracted from aliquots (100 mg) of frozen plants tissue using either Trizol reagent (Invitrogen) or an RNeasy Mini Prep Kit (Qiagen). After Trizol extraction, RNA was precipitated with isopropanol in the presence of 0.18 M tri-sodium citrate and 0.27 M NaCl. The precipitated RNA was pelleted by centrifugation at 12,000 x g for 10 min at 4°c. The supernatant was discarded and the pellet was washed twice with 75 % (v/v) of ethanol (prepared in diethyl pyrocarbonate (DEPC) treated water), briefly air dried, and then dissolved in 40 μl of distilled water and stored at -20°C until analysis. The concentration of the RNA was measured spectrophotometrically using a NanoDrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, USA; www.thermofisher.com).  The quality of the RNA was evaluated from the A260/280 ratio (values in the range of 1.8-2.0 indicate low protein contamination) and from the A260/230 ratio (values >2.0 indicate low polysaccharide contamination). The integrity of the RNA was checked by micro-capillary electrophoresis on RNA 6000 NanoChips using an Agilent-2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany; www.agilent.com). To remove genomic DNA, 10 μg of total RNA were digested with ribonuclease-free deoxyribonuclease at 37°C for 30 min using a Turbo DNA-free Kit (Ambion, Austin, USA; www.thermofisher.com). The absence of genomic DNA contamination was confirmed by PCR using ACTIN2 (At3g18780) primers based on intron sequences (Czechowski et al., 2004). The concentration and integrity of the RNA after deoxyribonuclease treatment were assessed as described above. For each sample, 5 µg of purified RNA was used for library construction and Illumina (www.illumina.com) single end sequencing by BGI Group (www.bgi.com). Quality control and barcode trimming were done by BGI while mapping and statistical analysis were performed using CLC Genomics Workbench (QIAGEN Aarhus A/S, www.qiagenbioinformatics.com).