BGISEQ-500 paired end sequencing; Transcription profiling of Populus euphratica roots and leaves in response to 150 mM NaCl
Roots were cut off and dipped immediately in RNAlater solution to prevent RNA degradation due to RNase activity. Apical 5 cm of main roots (primary root tip) and lateral root tips were separated, frozen in liquid Nitrogen and stored at -80 °C. Three leaves per plant were cut off, pooled, frozen in liquid Nitrogen and stored at -80 °C. Wild type Populus euphratica were multiplied by in vitro micropropagation (Müller et al., 2013). Six weeks old rooted plants were transferred into a hydroponics system in a green house. Plants were grown for 11 weeks in aerated hydroponics using Long-Ashton (LA) nutrient solution (Hewitt & Smith, 1974). Controlled conditions of 16 h photoperiod with 150 μmol×m-2×s-1 photosynthetically active radiation at 21–24 °C and 60–80% relative air humidity were maintained during the experiment. Control plants were grown in LA nutrient solution throughout the experiment. Salt treated plants were adapted to NaCl by increasing the concentration to 50 mM NaCl, after 1 day to 100 mM, and after 2 additional days to 150 mM. Plants were harvested after 2 and 12 days at 150 mM NaCl. Ethanol precipitation protocol and CTAB-pBIOZOL reagent was used for the extraction and purification of total RNA from plant tissue according to the manual instructions. Total RNA was qualified and quantified using a NanoDrop (Thermo Fisher Scientific, Waltham, USA) and Agilent 2100 bioanalyzer (Agilent, Santa Clara, USA). Oligo(dT)-attached magnetic beads were used to purified mRNA. Purified mRNA was fragmented into small pieces with fragment buffer at appropriate temperature. First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. A-Tailing Mix and RNA Index Adapters were added by incubating to end repair. The cDNA fragments obtained from the previous step were amplified by PCR, and products were purified by Ampure XP Beads, then dissolved in EB solution. The product was validated on the Agilent 2100 bioanalyzer for quality control. The double stranded PCR products from previous step were heated, denatured and circularized by the splint oligo sequence to get the final library. The single strand circle DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoball (DNB). DNBs were loaded into the patterned nanoarray.