title: RDA-seq (Next generation sequencing of Representational Difference Analysis products) of triticale leaves
design: RDA-seq (Next generation sequencing of Representational Difference Analysis products) of triticale leaves
library_name: Sample 5_p
library_construction_protocol: The plant material consisted of triticale leaves in two varieties (HEWO, MAGNAT) in two growth phases (vegetative, generative) and grown under two cultivation conditions (drought, control)  Seeds were sown in 3.7 L pots filled with a soil and sand mixture (1:3; v/v). Emerging seedlings underwent vernalization in cooling chambers for eight weeks at +4°C, under a photosynthetic photon flux density (PPFD) of 150 μmol m⁻² s⁻¹ and a photoperiod of 10 h light/14 h dark. After vernalization, plants at the two-leaf stage were transferred to greenhouse chambers. The greenhouse conditions were maintained at 25–30°C during the day and 15–20°C at night (±2°C), with a relative humidity of approximately 30–35%. Plants were supplemented with additional light, and the PPFD at the top leaf level was 200–250 μmol m⁻² s⁻¹, provided by high-pressure sodium lamps (400 W; Philips SON-T AGRO, Brussels, Belgium). Light intensity was measured using a QSPAR Quantum Sensor (Hansatech Instruments Ltd, Kings Lynn, England). Plants were irrigated weekly with a nutrient solution according to Hoagland (1948). Soil drought during the vegetative growth stage was induced when the plants reached the four-leaf stage, and during the generative stage when the flag leaf was fully developed. Over the course of seven days, water content in the pots was gradually reduced to 30% (approximately -0.507 MPa) by withholding irrigation, and this level was maintained for an additional two weeks. In control pots, water content was maintained at approximately 75% (about -0.006 MPa). Soil moisture was monitored daily by gravimetric method between 8:00 and 10:00 a.m., taking into account the plant biomass in the pots. GeneMATRIX Universal RNA Purification kit (EURx, Gdańsk, Poland) was used to isolate total RNA. RNA concentration was measured by means of a Qubit® 2.0 Fluorometer (Invitrogen, Waltham, MA, USA) and its quality and integrity were verified on BioAnalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) using Agilent RNA 6000 Nano Kit.  The first and the second strand of cDNA smART were obtained usin First Strand cDNA Synthesis Kit (EURx, Gdańsk, Poland) and Second Strand cDNA Synthesis Kit (abm, Richmond, BC, Canada), respectively. Representational difference analysis was performed on high quality cDNA according to Hubank and Schatz (1994) procedure with small modifications. Lodicules at the pre-flowering were used as testers while the lodicules early-flowering were selected as driver probes. The analysis was taken in three replicates for each variety. The tester:driver ratio in three subsequent rounds of subtractive hybridization increased from 1:50, and 1:400 to 1:200000. Differential products obtained in the third subtraction of the size range from 200 to 800 bp were sequenced. Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, USA) was used for library construction according to the manufacturer instructions.