title: Sample 14
organism: Kalanchoe pinnata
description: Protocols: K. daigremontiana and K. pinnata have different modes of plantlet formation; K. daigremontiana produces plantlets continuously under long-day conditions whereas K. pinnata leaves form plantlets upon detachment or ageing. Therefore, tissues of four different K. daigremontiana plantlets stages (S1, S2, S3, S4) were selected and harvested for the RNA-sequencing experiment. The control samples (Con) in K. daigremontiana were young with 1 to 2 cm leaf margins. For K. pinnata, tissues at the notches (N) of post-detachment leaves across the four time points (0 h, 4 h, 24 h, 48 h) were used. At 48 hours after leaves were detached from the plants, the midvein region of leaves was harvested. This mid-section (M) of leaves after 48 h post-detachment and undetached notches (0h) were used as control samples. Wild-type K. daigremontiana plants were grown in SANYO versatile environmental test chamber MLR-351 at 23˚C with a photoperiod of 16 hour/8 hours with 50 μmol m-2s-1 light and 60 % humidity. Wild-type K. pinnata plants were grown in Percival Scientific growth chamber AR-60L at 23˚C with a photoperiod of 8 hours/16 hours with 30 μmol m-2s-1 light and 60% humidity. The plants were grown in a mixture of 6 parts Levington® F2 Seed & Modular Compost (The Scotts Company, UK), 1 part Vermiculite V3 medium (Sinclair Pro, UK) and 1 part Perlite P35 standard (Sinclair Pro, UK). Four distinct stages of plantlet formation in wild-type K. daigremontiana were identified to include stages of plantlet initiation. Leaves exhibiting at least three of these stages of plantlet maturation along its leaf margin were carefully selected for use. The leaves were removed using a razor blade and 0.3 cm2 tissues at the leaf notches were harvested using the blade. The control samples were the whole margin of leaves of about 1-2cm long when measured from base to tip of each leaf. As for K. pinnata, four time points after leaf detachment (0 h, 4 h, 24 h, 48 h) were selected. 0.3 cm2 tissues at the leaf notches were harvested with a razor blade from unattached leaves (0 h) and leaves detached after 4 hours, 24 hours and 48 hours. No major morphological changes were present during these time points. All samples harvested were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Wild-type K. daigremontiana plants were grown in SANYO versatile environmental test chamber MLR-351 at 23˚C with a photoperiod of 16 hour/8 hours with 50 μmol m-2s-1 light and 60 % humidity. Wild-type K. pinnata plants were grown in Percival Scientific growth chamber AR-60L at 23˚C with a photoperiod of 8 hours/16 hours with 30 μmol m-2s-1 light and 60% humidity. The plants were grown in a mixture of 6 parts Levington® F2 Seed & Modular Compost (The Scotts Company, UK), 1 part Vermiculite V3 medium (Sinclair Pro, UK) and 1 part Perlite P35 standard (Sinclair Pro, UK). Total RNAs from each sample were extracted with Qiagen RNeasy Plant Mini Kit (Qiagen, UK) according to the manufacturer's protocol with modification. For K. daigremontiana samples, 600 μl of RLC buffer with 10 mg of Polyvinylpyrrolidone (PVP) with a molecular weight of 40,000 was used for a maximum of 100 mg tissue powder. For K. pinnata samples, 600 μl of RLC buffer with 10 mg of Polyethylene glycol (PEG) with a molecular weight of 40,000 was used for a maximum of 100 mg of tissue powder. The mixed solution of samples from K. daigremontiana or K. pinnata was then vortexed and incubated for 1 minute at 56°C and 80°C respectively before following the subsequent steps of the kit's protocol. Quality and integrity of the RNA samples were assessed using a 2200 TapeStation (Agilent Technologies) and then libraries generated using the TruSeq® Stranded mRNA assay (Illumina, Inc.) according to the manufacturer's protocol. Briefly, total RNA (0.1-4ug) was used as input material from which polyadenylated mRNA was purified using poly-T, oligo-attached, magnetic beads. The mRNA was then fragmented using divalent cations under elevated temperature and then reverse transcribed into first strand cDNA using random primers. Second strand cDNA was then synthesised using DNA Polymerase I and RNase H. Following a single 'A' base addition, adapters were ligated to the cDNA fragments, and the products then purified and enriched by PCR to create the final cDNA library.