Ta cloning vector

The porcine Coro1A gene was amplified from cDNA of H.parasuis infected Porcine alveolar macrophages (PAM) by PCR primed with the following primer pair: the forward primer 5′-TTT GAA TTC ATG AGC CGG CAG GTG GTC C-3′ and the reverse primer 5′-GGG GAA GCT TCT ACT TGG CCT GGA CTG TC-3′. The PCR product was cloned into the TA cloning vector and pET-30a plasmid, respectively (TakaRa Bio Inc., Otsu, Shiga, Japan). For sequencing the DNA of porcine Coro1A, the PCR product was inserted into the TA cloning vector (TakaRa), according to the instructions of the manufacturer. The ligation product was used to transform into E.coli DH5α Positive clones contaning the porcine Coro1A gene were sequenced. For the protein expression, the PCR product was inserted into the pET-30a vector, recombinant plasmids were transformed into E.coli BL₂₁ (DE3) cells to obtain the recombinant fusion protein designated His-rPoCoro1A. His-tagged recombinant protein purified under non-denaturin conditions (using Ni-NTA His Bind Resin).

Based on the Genethon human genetic linkage map [21 (link)] in the National Center for Biotechnology Information, microsatellite markers with heterozygosity less than 0.75 but above 0.5 located within 2 Mb of KIF21A were selected. Four microsatellite markers D12S1692, D12S331, D12S1048, and D12S1668 were selected in these families, and the reference sequence of the microsatellite markers was obtained from the National Center for Biotechnology Information database (NCBI; https://www.ncbi.nlm.nih.gov/). The primers for amplifying the microsatellite markers were listed in Supplementary File 2. The details of the PCR procedure were the same as those described in mutation analysis. The PCR product was cloned into the TA cloning vector (Takara Bio Inc., Tokyo, Japan) and sequenced directly by the ABI 300 Genetic Analyzer (Perkin-Elmer, America). Family pedigrees and locus haplotypes were generated using the Cyrillic 2.1 program (Cyrillic Software, Wallingford, Oxford-shire, UK), and reconfirmed by observation.

To evaluate mutations introduced in transfected transgenic tomato calli and shoots, a region of about 400 bp surrounding the target locus of gRNA was amplified by short-range PCR using a PCR kit as described above. In the Cel-1 assay, PCR products from transgenic plants were digested using a Surveyor® Mutation Detection Kit (IDT). In PCR-RFLP, the PCR products from transgenic tomato plants were digested with AccI. Mutated and the wild-type DNA fragments were separated by 2–2.5% agarose gel electrophoresis and stained by GelRed (Biotium). PCR amplicons were also cloned into the TA cloning vector (TaKaRa Bio) to determine their sequences by the Sanger method. Amplicon deep sequences for on- and off-targets mutation analyses were performed using Multiplex identifiers-labeled PCR^{17 (link)}. PCR products were subjected to Truseq on the MiSeq platform (Illumina). MiSeq data was analyzed using CLC Genomics Workbench software version 7.5.1 (CLC bio). All primers used for short-range PCRs used in the mutation analyses are listed in Supplementary Table 3.