GSM8075978: M82WL06hInternode rep5; Solanum lycopersicum; RNA-Seq
We prepared RNA-seq libraries according to the random-primer primed method published originally in (Townsley et al., 2015) with modifications from Kajala et al., 2021. Tissue samples were ground by hand and immediately incubated in 250 μL lysis buffer (LBB: 100 mM Tris-HCl, 1000 mM LiCl, 10 mM EDTA, 1% SD, 5 mM DTT, Antifoam A) supplemented with 5 μ l/ml β-mercaptoethanol before use. The samples were transferred to 2ml tubes and the tubes were beat 2 times for 1min at 30 Hz. The samples were incubated at room temperature for 10 min and spun down at 13,000 rpm for 10 min. One uL of 12.5 μM biotin-20nt-dT(Table 3.1) oligo was mixed into 200 μL of lysate in PCR tubes and incubated  at room temperature for 10 min. Then, we added 20 μL Biolabs® streptavidin beads per reaction for incubation. The mRNA from samples binds the oligo through the polyA tail, and the biotin in the oligo binds the streptavidin on the beads. This allows pulling down only the mRNA and washing the rest of the material off. So, the samples were separated into magnetic bead-bound mRNA and supernatant by 96 well Magwell® magnetic separator. The bead pellets were washed with 200 μl washing buffer series: washing buffer A (WBA; 10 mM Tris-HCl, 150 mM LiCl, 1 mM EDTA, 0.1% SDS), washing buffer B (WBB; 10 mM Tris-HCl, 150 mM LiCl,1mM EDTA), and low salt buffer LSB (20 mM Tris-HCl,150 mM NaCl, 1 mM EDTA). The mRNA was eluted in 16 uL Tris-HCl with 80C. These pulldown and washing steps were repeated a second time to remove non-specifically associating rRNA from the samples. We prepared RNA-seq libraries according to the random-primer primed method published originally in (Townsley et al., 2015) with modifications from Kajala et al., 2021. RNA fragmentation was carried out by preparing 10 μL mixtures of 1.5 μl 5X Thermo Scientific RT buffer, 0.5 μl Invitrogen ® random primers, 8 μl mRNA from the above elution. The mixtures were put into thermocycler for temperature-induced fragmentation and priming of 1st strand of cDNA (25°C 1 sec, 94°C 1.5 min, 4°C 5 min, 4°C hold).cDNA synthesis. We prepared first strand master mixture as following: 1.5 μL 5X Thermo Scientific RT buffer, 1.5 μL 0.1M DTT, 1 μL H2O, 0.5 μL 25mM dNTPs, 0.5 μL RevertAid H minus reverse transcriptase enzyme (Thermo Scientific). The mixture was added onto the samples and incubated in thermocycler: 25°C 10min, 42°C 50min, 50°C 10min, 70°C 10min, 4°C hold. The second strand synthesis, end repair, and A-tailing were performed by adding a 5 μL mixture to the first strand, consisting of 1.5 μL H2O, 0.4 μL 25mM dNTPs, 1 μL DNA polymerase I (Thermo Fisher), 0.1 μL RNaseH (NEB), 0.4 μL NEBNext® End Repair Module enzyme mix, 0.2 μL Taq, and 1.4 μ