Instrument: Illumina HiSeq 2000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: For transcriptome profiling, root tissue from three individual plants was pooled together to yield one root sample per treatment-species combination per day on each of the three sampling days, and shoot tissue from the same three plants was pooled together to yield one shoot sample per treatment-species combination per day on each of the three sampling days. RNAseq Library Preparation RNAseq libraries were prepared using the method described by (Kumar et al., 2012). Briefly, for each library, the pooled root or shoot samples were homogenized in lysis and binding buffer containing beta-mercaptoethanol. Tissue was disrupted using a mortar and pestle and a Mini-beadbeater-96 high-throughput cell disruptor (BioSpec Products, Bartlesville, OK). Oligo (dT)25-coated Dynabeads (Ambion, Foster City, CA) were then added to the lysate, and mixed for 10 minutes to allow oligo (dT) beads to hybridize to poly-adenylated mRNA. cDNA was then synthesized and digested to ~300 bp fragments using NEBNext DNA fragmentase enzyme mix (NEB, Beverly, MA, USA). Library fragments greater than 300 bp were purified using Ampure XP solid phase reverse immobilization (SPRI) magnetic beads (Agencourt Bioscience, Beverly, MA, USA). For each library, one of the ninety-six unique barcoded adapters described in Kumar et al (2012) was ligated to each library, enabling multiplexing. cDNA was quantified using a SYBR-green based method on a plate reader, libraries were pooled, and fragment size and quality was checked using a BioAnalyzer (Agilent Technologies, Santa Clara, CA). Translating Ribosome Affinity Purification (TRAP) To determine the effects of eCO2 on the translatome of shoot tissue, transgenic S. lycopersicum and S. pennellii expressing a FLAG-epitope on the ribosomal L18 (RPL18) under the 35S promoter (Ron et al., 2013) were grown in ambient and elevated [CO2] in the same conditions and chambers as described above. At 6 and 12 DAP, tissue was collected for the Translating Ribosome Affinity Purification (TRAP) protocol. Because the biomass of plant tissues in young seedlings was small, and because the TRAP populations were segregating, we pooled multiple plants (up to 15) together to ensure that adequate mRNA for library preparation could be collected via the ribosomal pulldown. The TRAP procedure has been described by (Zanetti et al., 2005). Briefly, tissues from transgenic plants expressing the FLAG epitope was ground in liquid nitrogen using a mortar and pestle, and homogenized in polysome extraction buffer. Samples were clarified by centrifugation and filtration through Miracloth, and clarified extract was added to magnetic beads that were coated in anti-flag antibody. Beads and extract were incubated at 4˚C using a nutator for 2 hours, and washed 6-7 times with wash buffer. Polysomes bound to magnetic beads were then frozen at -80°C until RNAseq library preparation. RNAseq libraries were prepared from TRAP polysomes using the method described by (Townsley et al., 2015). Lysis and binding buffer containing beta-mercaptoethanol was added to anti-flag magnetic beads and bound ribosomes, allowing elution of mRNA. mRNA was then hybridized to biotin-20nt-dT oligos, allowing pulldown of mRNA out of solution with the use of magnetic streptavidin beads. mRNA was then washed with three successive buffers, eluted off of beads at 80˚C, and a secondary wash was performed, by adding more biotin-20nt-dT oligos to the mRNA, repeating the bead pulldown, and the buffer washes. mRNA was then heat-fragmented, and first and second strand cDNA was synthesized. The optimal fragment size was selected using Ampure beads, and libraries were enriched using PCR.