GSM8076004: MMFR48hPith 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 μL. This mixture was incubated in a thermocycler following these temperature and time settings: 16°C for 20 minutes, 20°C for 20 minutes, 72°C for 20 minutes, and held at 4°C. Subsequently, the reaction mixture was left at room temperature for 5 minutes with a mix of Beckman® Ampure XP beads at 1.5 times the volume, followed by two washes with 200 μL of 80% ethanol each, until the beads were completely dry. Adapter ligation: The dry beads were mixed with 3 ul of annealed 1 μM universal adapter primers (8μL of 100μM each primer was combined with 784 μL of H2O and cooled through the program: 94°C 1min, (94°C 10sec) * 60 cycles -1°C /cycle, 20°C 1min, 4°C hold). Then, 7 μL ligase mixture (5 μL Enzymatics 2X Rapid T4 ligation buffer, 0.25 μL Biolabs® DNA ligase) was added for 15-min room temperature incubation. We added 10 μL of 50 mM EDTA and 25 μL Ampure XP Bead Resuspension buffer (15% PEG 8000, 2.5M NaCl) to each sample, washed with 80% EtOH twice, and eluted the ligated DNA fragments with 20-22 μL 10mM Tris.  Enrichment, indexing and quantification We used 10 μl of cDNA into enrichment mixed with 4 μL 5X Phusion HF (Thermo Fisher) buffer,2.6 μL H2O, 1 μL 2 μM PE1 primer, 1 μl 8 μM each EnrichS1 + S2 primers (Table 3.1), 0.2 μl 25mM dNTPs, and 0.2 μl Phusion Polymerase (Thermo Fisher). Each sample used 1 μl of appropriate unique indexed enrichment oligo (Townsley et al., 2015). The mixture was heated in thermocycler with the following program: 98°C 30 sec,  (98 °C 10 sec, 65 °C 30 sec, 72 °C 30 sec) *12 cycles, 72 °C 5 min, 10 °C hold. We cleaned the enriched libraries according to Bailey-Serres lab protocol: 1.1 volume of well-resuspended Ampure beads are mixed well with the samples, followed by washing twice by 200 μl of 80% EtOH and resuspending in 10 μl 10mM Tris pH 8.0.  One μl of the samples were used for quality check by Bioanalyzer High Sensitivity DNA Analysis kit, and we selected cleaned library of 200-350 bps based on the peaks. Libraries were pooled together for sequencing with the same target concentration. A final clean-up was done with 0.8 volumes of Ampure beads and elution to the final target volume.