Q5u hot start high fidelity dna polymerase

Genomic DNA was isolated from one-week plants using CTAB buffer (100 mM Tris–HCl pH8.0, 20 mM EDTA, 1.4 M NaCl, 2% CTAB, 1% PVP). For chop-PCR, 200 ng genomic DNA was digested with McrBC enzyme (New England Biolabs, M0272L) at 37°C for 2 h followed by heat-inactivation of enzyme at 65°C for 20 min. Both digested and undigested DNA was amplified by loci-specific primers.
For bisulfite sequencing of CEN180 insertion at ABI5 locus, 500 ng genomic DNA was bisulfite converted by using an EZ DNA Methylation-Gold kit (Zymo Research). The CEN180 insertion DNA was then amplified by Q5U Hot Start High-Fidelity DNA Polymerase (NEB #M0515). Gel-purified PCR products were cloned into pCR blunt vector (Thermo Fisher Scientific #K270020) and transformed into E. coli DH5α competent cells. At least 10 positive clones were sequenced (Genewiz) and analyzed with Kismeth (http://katahdin.mssm.edu/kismeth/revpage.pl).

For the bisulfite reaction, 1000 ng of genomic DNA was converted using a EZ DNA Methlyation-Lightning Kit (Zymo Research, USA) following the manufacturer’s instructions. Then 2 µL of converted products were amplified using Q5U Hot Start High-Fidelity DNA Polymerase (NEB, USA) according to the manufacturer’s instructions. PAS-specific PCR was performed in a total volume of 50 µL as follows: 30 s at 98 °C, 35 × (10 s at 98 °C, 30 s at 65 °C, 30 s at 72 °C), and 2 min at 72 °C. The 4qA-allele-specific primers were from Calandra et al. [23 (link)]. The obtained PCR products were purified using a FastPure Gel DNA Extraction Mini Kit (Vazyme, China). Purified PCR products were cloned into a pCE2 TA/Blunt-Zero vector using a 5 min TA/Blunt-Zero Cloning Kit (Vazyme, China) and transformed into Escherichia coli DH5α Electro-Cells.
At least 50 clones were chosen at random from each sample, and individual clones were sent for Sanger sequencing (Tsingke Biological Technology, China). Ten previously reported CpG sites were included as methylation markers. The methylation level for each site was calculated as ratio of methylated sites to total sites. The mean methylation level for the 10 CpG sites was calculated as the average level across the 10 sites. A schematic diagram of the 10 CpG sites is shown in Fig. 1B.

Genomic sites of interest were amplified into fragments of approximately 200 bp from genomic DNA samples using PrimeSTAR GXL DNA Polymerase (TaKaRa). See Supplementary Table 3 for the list of primers used and the average mapped ratio of corresponding primers. PCR products were purified using DNA Clean & Concentrator-25 (Zymo Research) for Sanger sequencing and targeted deep sequencing. Targeted deep sequencing libraries were prepared using the VAHTS Universal DNA Library Prep Kit for Illumina v.3 (Vazyme). Briefly, the PCR fragments were sequentially subjected to end repair, adapter ligation and then PCR amplification. DNA purification in library preparation was performed using Agencourt Ampure XP beads (Beckman Coulter), and library amplification was performed using Q5U Hot Start High-Fidelity DNA Polymerase (NEB) and VAHTS Multiplex Oligos Set 4/5 for Illumina (Vazyme). The final library was subjected to quantification using the Qubit dsDNA HS Assay Kit (Invitrogen) and sequenced using Illumina HiSeq X Ten.