Sybr green supermix kit

Assessment of virus replication was performed in Huh7 cells, A549 cells, or PBMC-derived monocytes. Cells were infected with the virus at an MOI of 1 unless stated otherwise and the incubated for 1 h at 37 °C. After incubation, the inoculum was removed and replaced with DMEM with 3% FBS and 1% penicillin-streptomycin. Cell supernatant and cell lysates were harvested at different time points. RNA extraction of cell lysates and supernatant were done using the RNeasy Mini Kit and QIAamp Viral RNA Mini Kit, respectively (Qiagen) according to the manufacturer’s instructions. Next, cDNA synthesis was performed using the qScript cDNA Synthesis Kit (Quantas Biosciences). Viral replication kinetics were measured by RT-qPCR using the SYBR Green Supermix Kit (Roche). All reactions were run on a Roche LightCycler 480, and data analysis was performed with LightCycler 480 software. The amount of viral RNA present in cell lysates was calculated as the gRNA:GAPDH ratio. The sfRNA:gRNA ratio was quantified according to a previously established method (41 (link)). The following primers were used to detect gRNA and sfRNA: gRNA forward, 5′-CCA​TGA​AGA​GAT​TCA​GAA-3′; sfRNA forward, 5′-GGA​CGT​TAA​AAG​AAG​TCA-3′; gRNA/sfRNA reverse, 5′-GCT​GCG​ATT​TGT​AAG​GG-3′.

Total RNA was extracted from sorted liver or bone marrow LSK cells using an E.Z.N.A.MicroElute Total RNA Kit. (OMEGA, Atlanta, USA). The RNA concentration was quantified using a Nanodrop 2000 spectrophotometer (BioTek, Winooski, VT, USA). cDNA was generated using a FastQuant RT Kit (Tiangen Biotech CO. Ltd., Beijing, China). Quantitative real-time polymerase chain reaction (qPCR) was performed using a SYBR Green Supermix kit (Roche, Basel, Switzerland). Gene-specific primers were used as follows: HES1 (encoding Hes family BHLH transcription factor 1): 5′-ACACCGGACAAACCAAAGAC-3′, 5′-ATGCCGGGAGCTATCTTTCT-3′; HES5 (Hes family BHLH transcription factor 5): 5′-CAAGGAGAAAAACCGACTGC-3′, 5′-GGCTTTGCTGTGTTTCAGGT-3′; Dll1 (delta like canonical notch ligand 1): 5′-CAGGACCTTCTTTCGCGTATG-3′, 5′-AAGGGGAATCGGATGGGGTT-3′; Dll4 (delta like canonical notch ligand 4): 5′-TTCCAGGCAACCTTCTCCGA-3′, 5′-ACTGCCGCTATTCTTGTCCC-3′; Jagged1 (jagged canonical notch ligand 1): 5′-CTACATACAGCATCTACATGC-3′, 5′-TCAGGCATGATAAACCCTAGC-3′; and Actb (beta actin): 5′-TGGAATCCTGTGGCATCCATGAAAC-3′, 5′-TAAAACGCAGCTCAGTAACAGTCCG-3′. The 2^−∆∆CT (cycle threshold) equation was used to calculate the relative expression of target genes against that of Actb [33 (link)].