Editseq software

Nucleic acid sequence of fliC from different Salmonella strains and non- Salmonella strains were acquired from the GenBank database. The predicted amino acid sequences were derived from the DNA sequences using Editseq software in the DNASTAR (version 7.1.0). MEGA 7 software (version 7. 0. 26) was used for phylogenetic analyses by Maximum Composite Likelihood (MCL) method. MegAlign software of DNASTAR was used for amino acid sequence similarity analysis by the Clustal W method. According to the above analysis, highly conserved region of FliC was chose and targeted, and the corresponding purified rFliC′ protein was subsequently used as immunogen to generate MAbs against Salmonella flagellin.

The Polymerase chain reaction (PCR) was used to amplify matched lymphocyte and tumour DNA from the patient primary cultures to determine mutational status and loss of heterozygosity of the PTEN gene. Standard PCR conditions were adapted for the analysis of PTEN exon 5 [34 (link)]. PCR products were column purified using Qiagen Quick PCR clean up kit (Qiagen, UK) and then directly sequenced using PTEN exon 5 sequencing primers (Table 2). The presence of mutations was determined by using EditSeq software (DNASTAR).
Loss of heterozygosity in PTEN was assessed using two LOH probes described in Dahia et al [34 (link)]; they included a dinucleotide repeat in PTEN intron 2 (AFMa086wg9) (probe 1) and a G/T sequence polymorphism in PTEN intron 8 (probe 2). The reverse primer for intron 2 was labelled with a JOE fluorescent dye enabling PCR fragment analysis, which was performed using Applied Biosystems ABI3130XL and data analysed using Gene Mapper software (Life Technologies). LOH was identified in the heterozygous tumour samples as a reduction in one of the peaks, which corresponded to one of the alleles of the PTEN gene. The G/T polymorphism was analysed using digestion with restriction enzyme HincII (New England Biosciences), where the polymorphism is differentially cleaved in heterozygous and homozygous individuals.

The mouse spermatogonia genomes were investigated before and after Tle6-KO was extracted, and PCR was conducted for the sequence near the Tle6–SgRNA target in a programmed thermal cycler (Bio-Rad, Hercules, CA, USA) (primer sequences are shown in Supplementary Table S1). The following PCR conditions were used: initial denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min, and then a 7 min final extension at 72 °C. Subsequently, a part of the amplified fragment was compared with the sequence published on NCBI to analyze the effect of the Tle6 knockout using EditSeq software (DNAStar, Madison, WI, USA). Another part of the fragment was ligated into the PMD18-T vector and amplified. We determined the sequence (primer sequences are shown in Supplementary Table S1), and then the SeqMan software (DNAStar) was used to detect the Tle6 knockout efficiency of the amplified sequence.

For RT-qPCR analysis, QuantiNova™ SYBR^® Green PCR Kit (Qiagen), ≤100 ng of cDNA, and 5 μM of forward and reverse primers were used according to the manufacturer’s instructions. EditSeq software (DNASTAR^®, Madison, WI, USA) and Primer 3 software version 4.1.0 [38 ] were used to design oligonucleotide primers that flank intron sequences of the target gene if possible. The forward and reverse primers for the following genes were used to perform RT-qPCR: MURF1, MAFBX, ITCH, CHIP, MDM2, USP19, USP14, A20, UCH-L1, CYLD, BAG3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) The primer sequences were shown in Table 2. RT-qPCR was performed using a LightCycler^® 480 (Roche Diagnostics, Mannheim, Germany) and LightCycler^® 480 software 1.5.0 SP3 with the following protocol: 2 min at 95 °C, 40 cycles of 5 s at 95 °C and 10 s at 60 °C. Subsequently, the melting curves were recorded, and the correct size of the amplicons was determined by agarose gel electrophoresis. Threshold cycle (C_T) values were set within the exponential phase of the RT-qPCR. Data were normalized to GAPDH expression, and 2^−ΔΔCT values were used to calculate the relative expression levels of the genes.

The complete nucleotide sequence of GX2020-019 was aligned with 49 reference strains of FAdV-A to E available from the GenBank database using the ClustalW multiple alignment algorithm (shown in Table 1). A phylogenetic tree was created by neighbor-joining analysis with 500 replicates for bootstrapping, and evolutionary distances were calculated using the maximum composite likelihood method through MEGA 11 software (version 11, Molecular Evolutionary Genetic Analysis, New Zealand). MegAlign software (version 7.1, DNASTAR, United States) was utilized for a full-genome similarity comparison of GX2020-019 with pathogenic strains (GX2019-004, SD1601, JS07, SCDY, SD1511, and HLJFAd15 strains), nonpathogenic strains (ON1, KR5, and B1-7 strains) of FAdV-C, and reference strains of FAdV-A, B, D, and E. The major structural protein genes and ORFs of GX2020-019 were translated into amino acid sequences using EditSeq software (version 7.1, DNASTAR, United States). These sequences were compared with the sequences of FAdV-4 pathogenic strains (MX-SHP95, GX2019-004, SD1601, JS07, SCDY, SD1511, and HLJFAd15 strains) and nonpathogenic strains (ON1, KR5, and B1-7 strains) using MegAlign software (version 7.1, DNASTAR, United States).