Sanger method

For PCR amplification of the complete cDNA of the tisa-cel vector region containing the CAR, primers were designed by educated guess to bind in front of the CAR’s cDNA (EF1alpha-Forward 5′-GGTGGAGACTGAAGTTAGGCC) and behind the CAR’s cDNA in reverse orientation (wPRE-Reverse 5′-GCAATGCCCCAACCAGTG). The obtained PCR fragment was sequenced using the standard Sanger method (Eurofins Genomics, Ebersberg, Germany) with the indicated forward primer, and subsequently an additional internal primer (5′-CAGCCATTTACTACTGTGCCAA) was designed based on the first obtained sequence.
Primers and probes for three different amplicons (U, T, A), all located in the FMC regions of the CAR construct, were designed using PrimerExpress_3.0.1 (ThermoFischer, Kandel, Germany). One amplicon was universal (U) for both tisa-cel and axi-cel, whereas the other two were designed to be tisa-cel (T) and axi-cel (A) specific by locating the reverse prime in the different linkers present in the two vector constructs (Figure 1a). The universal primer/probe combination (U) is available from Bio-Rad (Foster City, CA, USA) as an Expert Design Assay (cat. number dEXD88164642).

For all environmental isolates in this study, cyp51A was PCR-amplified using previously designed primers (Mellado et al. 2001 (link)). PCRs were performed with the Q5 Hot Start High-Fidelity 2× Master Mix (New England Biolabs) with 100 ng of genomic DNA, 0.5 μM forward primer 5′-CGGGCTGGAGATACTATGGCT-3′ and 0.5 μM reverse primer 5′-GTATAATACACCTATTCCGATCACACC-3′ in 20 μl reactions. PCRs were performed at 98°C for 2 min followed by 30 cycles of 98°C for 15 s, 62°C for 15 s, and 72°C for 2:30 min, followed by a final extension at 72°C for 5 min. Amplicons were sequenced by the Sanger method (Eurofins genomics, USA) using primers 5′-GCATTCTGAAACACGTGCGTAG-3′, 5′-GTCTCCTCGAAATGGTGCCG-3′, and 5′-CGTTCCAAACTCACGGCTGA-3′. Promoter sequences were aligned to A1163 genomic sequence v43 from Ensembl. Coding sequences were translated to protein sequences and aligned to the Cyp51A protein of A. fumigatus A1163 (GenBank accession number EDP50065). Sequence analysis was performed using Geneious v11.1.5 (Biomatters, Auckland, NZ).

Total genomic DNA was extracted from HSV-1 ANG and ANG path virions using the QiaAmp Mini DNA Kit (Qiagen, Louisville, KY, USA). Full-length nucleotide sequences of UL44, UL27, US6, UL22, UL1, and UL45 genes, encoding gC, gB, gD, gH, gL, and UL45p, respectively, were obtained using flanking primers based on HSV-1 KOS and 17 strains. Internal primers based on the generated sequences were also used to obtain the full-length gene when necessary. Sequencing was performed using the Sanger method (Eurofins Genomic LLC, Loisville, KY, USA). Sequences were proofread and assembled using Vector NTI (Invitrogen, Carlsbad, CA, USA) and/or SnapGene (Dotmatics, Boston, MA, USA). Nucleotide and amino acid sequences were compared using Blast software and databases (NCBI, Bethesda, MD, USA).
The DNA sequences of HSV-1 ANG gB, gD, gH, gL, gC, and UL45 and HSV-1 ANG path gH, gL, gC, and UL45 were deposited to GenBank under the respective accession numbers, which are as follows: OQ263027, OQ263028, OQ759608, OQ263025, OQ263031, OQ759609, OQ263026, OQ263030, and OQ263032.

The presence of Asaia was investigated in 20 female and 20 male adults from the three C. capitata populations. Asaia detection was performed by PCR using specific oligonucleotides targeting the 270-bps fragment of the 16S rRNA gene described in Favia et al. [8 (link)]. A total of 50 ng of genomic DNA were used in PCR reaction containing 1X Buffer, 0.25 mM dNTPs, 0.9 U DreamTaq Polymerase (Thermo Scientific, Waltham, MA, USA) and 200 nM of Asafor (5′-GCGCGTAGGCGGTTTACAC-3′) and Asarev (5′-AGCGTCAGTAATGAGCCAGGTT-3′) oligonucleotides. The amplification protocol included: initial denaturation at 95 °C for 3 min, followed by 30 cycles consisting of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s and concluding with a final extension step of 10 min at 72 °C. The PCR products were electrophoresed on a 1% agarose gel to determine the presence and size of the amplified DNA. The amplicons were purified and sequenced by the mean of the Sanger method (Eurofins Genomics, Ebersberg, Germany). The 16S RNA sequences were analyzed by BLASTN (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 1 May 2022).

Coding sequences of Marwari horse mature IL-4 and IL-10 gene [16 ] were subcloned into Sac I and Hind III (Thermo Scientific FastDigest, UK) restricted prokaryotic expression vector pQE30 (Qiagen, Germany). The recombinant pQE30 vectors with respective IL-4 and IL-10 insert were transformed in Escherichia coli M15 competent cells prepared by Z-competent™ E. coli transformation kit (Zymo Research, USA). Positive clones were selected on Luria Bertani agar (LBA) plates supplemented with kanamycin (30 μg/ml) and ampicillin (50 μg/ml) (HiMedia, India) and screened by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, 10 colonies were randomly picked and were grown in 5 ml of LB broth at 37°C at 170 rpm. At mid-log phase (approximate O.D₆₀₀ 0.6), 1 mM isopropyl-β-D-thiogalactoside (IPTG) (Promega, USA) was added to culture and was further incubated for 3-6 h. Samples from bacterial cells were prepared as per standard protocol [17 ] and run on 12% SDS-PAGE. The gel was stained by Coomassie brilliant blue dye and protein band was visualized by destaining. For further confirmation of insert, two positive recombinant clones were sequenced by Sanger method (Eurofins genomics, India) and sequences were verified by NCBI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) program.