NextSeq 500 paired end sequencing; Expression profiles of bladder and stalk cell enriched epidermal peels from Chenopdium quinoa
Expression profiles of bladder and stalk cell enriched epidermal peels from Chenopdium quinoa
Detached leaves were submerged in BSM (to avoid dessication of the stalk cells) and then lightly brushed with a fine paintbrush (diameter 2 mm) as above described for petioles. Subsequently, the leaves were quickly removed from the BSM and the abaxial epidermis was removed using fine tweezers. The epidermal peel was then quickly placed in RNAlater Stabilization Solution (Thermo Fisher Scientific) for RNA sequencing or floated into covered petri dish containing BSM with 5 μg mL-1 FDA for 15 min for FDA staining. As a negative control, leaves still attached to the plants were forcefully brushed (i.e. hard brushing) and then the epidermal peels were collected 30 min later (to ensure that any viable stalk cells would dry) as above described. Intact segments of non-brushed leaves were used as positive controls. Quinoa plants were grown from seeds in 15 cm diameter pots filled with standard potting mix under temperature-controlled glasshouse conditions. The standard potting mix was consisted of 90% composted pine bark; 5% coco peat; 5% coarse sand; gypsum at 1 kg/m3; dolomite at 6 kg/m3; ferrous sulphate at 1.5 kg/m3; Osmoform Pre-mix at 1.25 kg/m3 and controlled-release fertilizer, Scotts Pro at 3 kg/m3. Day/night temperatures were 20 °C and 15.5 °C; mean humidity 74% and day length 16 h (incandescent lights were set at 6.00 to 9.00 and 16.00 to 22.00 hrs to give the day length hours). Ten seeds were sown in each pot and germinating seedlings there then thinned to 4 uniform plants per pot a few days 14 days after sowing. Epidermal peels were removed from the RNAlater Stabilization Solution (Thermo Fisher Scientific) and excess RNAlater was bloted away quickly and tissue was frozen in liquid nitrogen immediately for RNA isolation. Total RNA was isolated using the the NucleoSpin RNA Plant Kit (Macherey-Nagel) according to the manual. In brief, the plant tissue was frozen with liquid nitrogen and homogenzied with mortar and pestle manually. 50mg of the frozen plant powder were mixed strongly with 350µl of the lysis buffer RA1 containing TCEP (1:100). The lysate was transferred to the NucleoSpin Filters and centrifuged for 1 min at 11,000 g. The filtrate was mixed with 350 µl 70% ethanol and transferred to the NucleoSpin RNA plant Column and centrifuged for 1 min at 11,000 g. The washing steps were performed following the manufacturer's instructions. RNA was eluted in 33 µl RNase free water that was incubated on the membrane twice for 1 min. Quality control measurements were performed on an Experion TM automated electrophoresis station (Bio-Rad Hercules, California, U.S.A.) and the concentration was determined using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, U.S.A.). Library preparation and RNAseq were carried out as described in the Illumina TruSeq Stranded mRNA Sample Preparation Guide, the Illumina NextSeq 500 System Guide (Illumina, Inc., San Diego, CA, USA), and the KAPA Library Quantification Kit - Illumina/ABI Prism User Guide (Kapa Biosystems, Inc., Woburn, MA, USA). In brief, 200 ng of total RNA was used for purifying the poly-A containing mRNA molecules using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented to an average insert size of 200-400 bases using divalent cations under elevated temperature (94°C for 4 minutes). Next, the cleaved RNA fragments were reverse transcribed into first strand cDNA using reverse transcriptase and random hexamer primers. Actinomycin D was added to improve strand specificity by preventing spurious DNA-dependent synthesis. Blunt-ended second strand cDNA was synthesized using DNA Polymerase I, RNase H and dUTP nucleotides. The incorporation of dUTP, in place of dTTP, quenched the second strand during the later PCR amplification, because the polymerase does not incorporate past this nucleotide. The resulting cDNA fragments were adenylated at the 3' ends, the indexing adapters were ligated and subsequently specific cDNA libraries were created by PCR enrichment. The libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit.