Chapter 3 90 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. 3.5.4.3 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. 3.5.4.4 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.
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