Kalanchoe blossfeldiana_LP9_r2_dusk_rna
RNA was extracted from young (9th leaf pair from the bottom) and mature (3rd leaf pair from the bottom) leaves, at dusk. Samples were flash frozen in liquid nitrogen, then 100 mg of leaf tissue was ground in liquid nitrogen. The samples were the same as those used in chapter one for metabolite analysis. Ground tissue was combined with 900 l of pre-heated (65C) extraction buffer: 3% Hexadecyltrimethylammonium bromide (CTAB) (w/v), 100 mM Tris-HCl (pH 8), 25 mM EDTA (pH 8), 2 M NaCl, 3% polyvinylpyrrolidone (PVP-40) (w/v), 0.5g/L spermidine and 4% -mercaptoethanol added just prior to extraction and incubated at 65C for ten minutes while shaking. Exactly 900 l of chloroform: isoamyl alcohol (24:1) was added and mixed by inversion for ten minutes. Samples were centrifuged at max speed for ten minutes at 4C, with supernatants transferred to fresh tubes with an equal volume of chloroform: isoamyl alcohol (24:1), then incubated at 4C for ten minutes while inverting. The upper phase was discarded and two times volume of ethanol added, followed by freezing at -20C for two hours and centrifuged at 15,000 g for 30 minutes.  The supernatant was discarded and 1 ml lithium chloride added, followed by two hours at -20C and centrifuged at 15,000 g for 30 minutes. The supernatant was discarded and the RNA pellet was resuspended in 100 l of DEPC water, with gentle shaking at 65C for ten minutes. Care was taken throughout to avoid RNAse contamination.  Samples were then DNase treated with an Invitrogen DNase kit, as described by its manual. Short read RNA library preparation and sequencing of K. blossfeldiana was carried out by Novogene (Beijing, China). Three biological replicates were collected and sequenced from each leaf age group, with samples taken at dusk. A total of 119 million PE reads, and 35.8 Gb were sequenced on an Illumina NovaSeq 6000, with a range of 15-22 million reads per sample.