Quantstudio thermo cycler

For transient downregulation of GLB1, a total of 30,000 control or 50,000 senescent A549 and SK‐MEL‐103 cells were plated per well in a 24‐well plate. The next day, cells were transfected with TriFECTa^® Kit DsiRNA Duplex siRNAs (Integrated DNA Technologies) hs.Ri.GLB1.13.1 (siRNA1), hs.Ri.GLB1.13.3 (siRNA2) or scrambled siRNA, using Lipotectamine RNAiMAX Reagent (Thermo Fisher Scientific) as per manufacturer's instructions. After 48 hr, RNA was extracted using the RNeasy Mini Kit (Qiagen) and cDNA was synthesized with the High‐Capacity RNA‐to‐cDNA™ Kit (Thermo Fisher Scientific).
Gene expression of GLB1 and ACTB genes was measured by quantitative real‐time PCR performed on a QuantStudio thermocycler (Applied Biosystems) following Luna^® Universal qPCR Master Mix (New England Biolabs) protocol and amplification parameters, and using predesigned KiCqStart^® SYBR^® Green Primers H_GLB1_1 and H_ACTB_1 pairs (Sigma‐Aldrich). Relative quantification was carried out using 2−ΔΔCt methodology.

All patients were confirmed with SARS-Cov-2 using routine RT-PCR methodology, with two sets: either Allplex® 2019-nCoV assay (Seegene, Seoul, South Korea) with Microlab NIMBUS® extractor (Hamilton Bonaduz AG, Rapperswil-Jona, Switzerland) and CFX96® thermocycler (Bio-Rad laboratories, Hercules, California, United States of America); or RealStar® SARS-CoV-2 RT-PCR Kit 1.0 assay (Altona Diagnostics, Hamburg, Germany) with QIAsymphony SP® extractor (QIAGEN, Hilden, Germany) and QuantStudio® thermocycler (Thermo Fisher Scientific, Waltham, Massachusetts, United States of America). RT-PCR was performed on nasopharyngeal swabs or on distal bronchial samples.

Total RNA of the cotton mealybugs was extracted using TRIzol reagent, and 1 μg of RNA was reverse-transcribed using HiScript III 1^st Strand cDNA Synthesis Kit (Vazyme Biotech). We designed gene-specific primers based on the sequence of 16S rRNA of T. phenacola PSOL for endosymbiont quantification, with the host’s Actin selected as the reference gene (Table S3). Quantitative reverse transcription was performed on a QuantStudio thermocycler (Thermo Fisher Scientific) in 20-μl reaction volumes containing 10 μl of HiScript III RT SuperMix (Vazyme), 1 μl of 100 ng/μl cDNA, and 1 μl of 50 ng/μl primers. The thermocycling program was as follows: initial denaturation, 95°C for 10 min; 40 cycles of denaturation (95°C for 15 s) and annealing (60°C for 30 s). The data were analyzed using the 2^−ΔΔC method [57 (link)]. These experiments were conducted with at least three independent biological replicates.

RT-PCR and quantitative real-time PCR (qPCR) were performed using previously reported protocols [39 (link)]. Total RNA was extracted by using Direct-zol RNA kit and following the manufacturer’s protocol (Zymo Research). cDNA pool was synthesized by qScript cDNA Supermix (Quantabio) using 2 μg of total RNA. cDNA from clone Xi8 and Xa3 RNA was further used for qPCR to analyze reactivated mouse MeCP2. qPCR was performed using SsoAdvanced Universal SYBR green Supermix (BioRad). The primer sets were β-actin (forward, 5′-AGAGCTACGAGCTGCCTGAC-3′; reverse, 5′-AGCACTGTGTTGGCGTACAG-3), mouse MeCP2 (forward, 5′-CATACATAGGTCCCCGGTCA-3′; reverse, 5′-CAGGCAAAGCAGAAACATCA-3′), mouse Xist (forward 5′-GGTTCTCTCTCCAGAAGCTAGGAAAG-3′; reverse, 5′-GGTAGATGGCATTGTGTATTATATGG-3′), and mouse Rnf12 (forward, 5′-CTTGGATCGGGAAGAGGCTT-3′; reverse, 5′-TTCACCTGGGGTGCCCAGCA-3′). qPCR was performed using QuantStudio thermocycler (Thermo Fisher) with the following conditions: 95 °C for 5 min and followed by 40 cycles of 95 °C 10 s, 60 °C 1 min. Relative quantity (RQ) was determined after normalized ΔΔCt was calculated.