Longamp taq

Total RNA was extracted from the leaf samples using a RNeasy Plant Mini Kit (Qiagen, Valencia, CA, United States, product No. 74903). After removal of the relic using RNase-free DNase (Qiagen), the quality of the RNA sample was assessed using 1% agarose gel electrophoresis, a NanoDrop spectrophotometer (ThermoFisher Scientific, Wilmington, DE, United States), and an Agilent 2,100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, United States). The RNA was then reverse-transcribed to cDNA according to the protocol by Oxford Nanopore Technologies (Jain et al., 2016 (link)). The sequencing libraries were created using a preparation kit (Ultra-Long, Grandomics, Wuhan, China) by fragmenting the cDNA and adding PCR adapters to both ends. After PCR amplification (14 cycles) with LongAmp Taq (NEB, New England Biolabs LTD., Beijing, China), the ONT adaptors were ligated onto the PCR products using T4 DNA ligase (NEB). The ligated PCR products were sequenced on a MinION Mk1B sequencer (Oxford Nanopore, Oxford, United Kingdom). All sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) under the data have been released from the SRA database since November 23, 2022.

Genomic DNA was isolated from each well of a confluent 24- or 96-well plate as follows: cells were incubated for 2 h at 55°C in 20 or 40 μl of lysis buffer (0.45% NP40, 0.45% Tween20, 1× NEB LongAmp PCR buffer) and subsequently heated to 95°C (10 min). One to two microlitres of this lysate was used in a 10 μl PCR reaction. PCR reactions comprised 0.2 μl DMSO (100% v/v, Sigma), 0.3 μl dNTPs (10 mM, Thermo Fisher Scientific), 2.0 μl LongAMP buffer (5× NEB), 0.4 μl LongAMP Taq (NEB) and 12 pmol of each primer. Thermal cycling was performed using the following conditions: 1 cycle 94°C for 3 min; 40 cycles 94°C for 15 s, 60°C for 30 s, 65°C for 2 min; followed by final extension at 65°C for 10 min.
For each targeted locus, two sets of genotyping primers spanning the junction of genomic sequences and targeting vector were used (left and right arms). Gene-specific primers were designed outside the 5′ and 3′ homology arms and were used in combination with primers in the knock-in cassette (either CAG-LUC-2A-GFP-IRES-BSD for targeting Rosa26 and AAVS1 loci, or Ef1α-Puromycin for the knockout experiments). To identify NHEJ-based indel formation on the second, non-targeted alleles, the region flanking the sgRNA target sites (500-600 bp) was amplified using PCR with gene-specific primers and directly assessed by Sanger sequencing. Sequences of all primers are provided (Table S2).

The total RNA was extracted from the fruits using an improved CTAB-based protocol (Leh et al., 2019 (link)). One microgram of total RNA with high purity and integrity (RIN > 8.5) was used for the library construction. A cDNA-PCR sequencing kit (SQK-PCS109) was used referring to the protocol of ONT. Briefly, full-length cDNAs were enriched by the template-switching activity of the reverse transcriptase. Specific PCR adapters were added at both ends of the first-strand cDNA. After 14 circles of PCR amplification using the LongAmp Taq (NEB, USA) enzyme, ONT adaptors were ligated to the amplified products. Afterward, Agencourt XP beads were used for DNA purification according to the manufacturer's protocol. The final 12 cDNA libraries were loaded into the FLO-MIN109 flowcells and sequenced on the PromethION platform at Biomarker Technology Company (Beijing, China).

In order to further study the loss of T<>T from the mitochondrial genome noted by DDIP-seq, we performed “short range” qPCR of mtDNA damage in HaCaTs, using UVC as an effective source of T<>T. Using the method based on that of Santos et al. [63 (link)], total genomic DNA from UVC irradiated HaCaTs underwent PCR (Mastercycler Pro; Eppendorf, Hamburg, Germany) using primers specific for a 221 bp region spanning the Cytb and ND6 genes, and using LongAmp Taq (New England Biolabs, Ipswich, MA, USA). The PCR products were quantified in a Synergy 2 microplate reader (BioTek, Winooski, VT, USA), based upon PicoGreen fluorescence. Lesion frequency/10 kb was calculated, according the formula reported elsewhere [64 ].

Pyrosequencing was used to test allelic expression imbalance of PDSS2 and SOBP. Total RNA was purified with DNase I (NEB) and minus-RT (not reverse transcribed) PCR products were used as negative controls. Two SNPs in PDSS2 (ss666793773 in exon 8 and ss189596174 in exon 6) and two SNPs in SOBP (ss666793686 and ss666793687 in exon 6) were designed for PCR amplification. The pyrosequencing PCR assays contained 40 ng cDNA (or gDNA), 1× PCR buffer, 200 µM dNTP, 400 nM of each primer, 2 IU LongAmp Taq (NEB), H₂O was added to give a final volume of 25 µl. A touchdown PCR protocol was used for the pyrosequencing SNP genotyping test including 94°C for 5 min, 10 cycles of 94°C, 63°C (−1.0°C/cycle), and 65°C for 30 s each, followed by 30 cycles of 94°C, 53°C, and 65°C for 30 s each, and a final extension at 65°C for 5 min. The PCR product was analyzed by 2% agarose gel electrophoresis and used for pyrosequencing according to standard protocol of PyroMark ID (Qiagen). The relative proportion of each allele was obtained using the AQ analysis mode (allele quantification). The details of all primer sequences were given in Table S5.

Genotyping of PDSS2(-103C-G) (ss666793747) and ss666793770 was performed using pyrosequencing. The pyrosequencing PCR assays contained 40 ng gDNA, 1× PCR buffer, 1× Q-solution (Qiagen), 200 µM dNTP, 400 nM of each primer, 2 IU LongAmp Taq (NEB), and H₂O was added to achieve a final volume of 25 µl. A touchdown PCR protocol was used, including 94°C for 5 min, 10 cycles of 94°C, 63°C (−1.0°C/cycle), and 65°C for 30 s each, followed by 30 cycles of 94°C, 53°C, and 65°C for 30 s each, and a final extension at 65°C for 5 min. The PCR product was analyzed by 2% agarose gel electrophoresis and used for pyrosequencing according to standard protocol of PyroMark ID (Qiagen).

DNA LoBind tubes (Eppendorf) were used. PCR was performed with tailed-end primers using the same conditions as for amplicons produced for Sanger sequencing, to generate amplicons for ONT sequencing. PCR amplicons were barcoded using LongAmp Taq (New England BioLabs). The ends of pooled DNA fragments were repaired using the NEBNext End repair/dA-tailing Module (New England BioLabs). Sequencing adaptors were added using the 1D- Ligation Sequencing Kit (ONT). All reactions were performed according to the manufacturer’s instructions. DNA was purified at all steps using AMPure XP beads (Agencourt) employing a 0.8X to 1X beads to sample ratio. DNA was quantified with a Qubit fluorometer at all steps. Sequencing libraries were loaded on a primed SpotON Flow Cell (R9.4) (ONT). Runs were performed using the MinKNOW GUI at default settings for up to 24 hours (ONT).