T100 pcr system

The purified myxobacterial isolates were inoculated on VY/4 medium for morphological observation. For the phylogenetic analysis, the 16S rRNA gene was amplified using the primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) (Weisburg et al., 1991 (link)). The PCR amplification was conducted in a T100™ PCR system (Bio-Rad, Hercules, CA, United States) using EasyTaq DNA polymerase (Transgene, China) with following conditions: 94°C for 5 min, followed by 35 cycles at 94°C for 15 s, 58°C for 30 s, and 72°C for 2 min, and a final extension at 72°C for 5 min. PCR amplification products were detected using 1% agarose gel electrophoresis analysis and sequenced by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China). The similarity search of the 16S rRNA gene was performed using EzBioCloud database.¹

HEK293T/hACE2 cells transfected with constructs encoding hAPP, and HEK293T/hAPP cells transfected with constructs encoding hACE2, as well as SH-SY5Y, SH-SY5Y/APP695wt, and SH-SY5Y/KD-APP, were infected with the GFP-labeled pseudovirus at a concentration of 10¹⁰/mL and incubated for 72 h at 37 °C. Meanwhile, AAV/Vector mice, AAV-APP695wt mice, and AAV-APP695wt/AY51 mice were infected with the GFP-labeled pseudovirus at 10¹⁰/mL for 7 days. Vero E6 cells were plated at a density of 3 × 10⁴ cells/well in 96-well plates and were incubated for 24 h. These cells were then infected with SARS-CoV-2 at an MOI of 0.05 in the presence of various concentrations of AY51 (2.5, 5, 10, 20, 40, and 80 μM). The amount of pseudovirus present in the cell lysate was quantified using qRT-PCR.
Total RNA was extracted from either the cell lysates or homogenized brain tissues using TRIzol reagent (Catalog#15596026, Invitrogen) as per the manufacturer’s instructions. The extracted RNA was then reverse-transcribed into cDNA using the Prime Script II 1st Strand cDNA Synthesis Kit (Catalog#6210A, TaKaRa) on the T100 PCR system (Bio-Rad, USA). The resulting cDNA was amplified using specific primers (listed in Table S1) and the TB Green^® Fast qPCR Mix (Catalog#RR430A; TaKaRa). The relative expression levels of the pseudovirus were measured using the CFX Connect Real-Time System (Bio-Rad, USA).

RNA isolation was performed using RNeasy^® Mini Kit (Qiagen, Hilden, Germany), according to standard protocol. Reverse transcription was conducted with HiScript III 1st Strand cDNA Synthesis Kit (Nanjing Vazyme Biotech Co. Nanjing, China) using random hexamers and primer containing 50 µM oligio (dt). The process was carried out at 25°C for 10 min, 48°C for 30 min, and 95°C for 5 min on a T100 PCR system (Bio-Rad Laboratories, Hercules, CA, USA).

Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer’s instructions, and then was synthetized into cDNA with an RT reagent Kit (code number is RR047A; Takara, Dalian, China) for qRT-PCR and 1st Strand cDNA Synthesis Kit for RT-PCR (code number is 6110A; Takara, Dalian, China). All qRT–PCR assays were performed with the Premix Ex TaqKit (code number is RR420A; TaKaRa, Dalian, China) in a CFX Connect Real-time PCR System (BIO-RAD, Hercules, CA, USA). RT-PCR assays were performed with the Ex Taq DNA Polymerase (code number is RR001A; TaKaRa, Dalian, China) in a T100 PCR System (BIO-RAD, Hercules, CA, USA). All of the primers used here are listed in Supplementary Table S1. qRT-PCR analysis was performed as previously described [52 (link)] with three independent biological replicates.

RNA extraction methods have been described in detail in previous studies (Kong et al., 2015 (link)). Total RNA was reverse transcribed using Hifair™ 1st Strand cDNA Synthesis SuperMix for PCR (YEASEN, China). PCR was run on the T100 PCR system (Bio-Rad, United States) using PrimerStarMAX DNA Polymerase (TaKaRa Bio, Japan) according to the manufacturer’s protocol. The primers were synthesized by Tsingke Company (Target exon 4 – exon 6: 5′-CTAGAGAACCCACTGCTTAC, 3′-TAGAAGGCACAGTCGAGG). The purified PCR products were separated and purified via 2% agarose and sequenced as described above.