Cfx384

Genomic DNA was isolated by ethanol precipitation and RNA was isolated from tissue using RNeasy Mini kits (Qiagen). Samples were run using the Luna Universal One-Step RT-qPCR Kit (New England Biolabs) on a CFX384 (Bio-Rad).

RNA was isolated from intact human islets using Quick-RNA Microprep kit (Zymo). Briefly, after indicated treatments, medium was removed from islets and RNA lysis buffer (with β-mercaptoethanol) was added to cells in 1.5 mL tubes, vortexed, and then transferred to -80°C for storage. Samples were processed according to the kit manufacturer’s instructions, including on-column digestion with RNase-free DNase. RNA concentration was measured using a Nanodrop spectrophotometer and verified to have A_260/280 ratios >2.0. 600 ng of RNA was converted into cDNA using the iScript cDNA synthesis kit (Bio-Rad) following manufacturer instructions and the resulting cDNA was diluted 10-fold with water. One μl of diluted cDNA was used in 10 μl qPCR reactions using 2X SYBR Bio-Rad master mix and 250 nM of each primer. Reactions were run in 384-well format on a CFX384 (Bio-Rad) or QuantStudio 5 (Thermo). qPCR data was analyzed using CFX Manager (Bio-Rad) with 18S RNA as the reference gene. Relative expression was calculated by the 2^-ΔΔCt method.

Total RNA was extracted from tissues and cell cultures using Trizol Reagent (Ambion) and treated with a RQ1 RNase-free DNase (Promega). One µg of RNA was used to generate cDNA with miScript II RT kit (Qiagen) for snRNA analyses and Superscript III (Invitrogen) for the other genes. Quantitative real-time PCR was performed in triplicate using the primers listed in Supplemental Table 2 with SYBR Green ROX mix (Thermo Scientific) on either Applied Biosystems 7500 fast system or BioRad CFX384. The normalized expression levels were calculated according to the ΔΔCt method. The snRNA, 5 S, 5.8 S, Rpl13a, Ppia, Gapdh, Myh4 and Z⁺Agrn primers and their analyses have been previously described^{43 (link)}. Additional primers have been designed using the free primer design tools from eurofins (eurofinsgenomics.eu) and validated according to the MIQE guidelines.

RNA was extracted from liquid nitrogen frozen mycelium using TRIzol Reagent (Thermo Fisher Scientific). 2 μg of total RNA was then subjected to DNase I digestion (Thermo Fisher Scientific). cDNA was amplified using iScript™ cDNA Synthesis Kit (Bio‐Rad) and LunaScript® RT SuperMix Kit (NEB). cDNA was diluted 1:10 and used for qRT‐PCR (Bio‐Rad CFX384).

Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Milan, Italy) and converted into cDNA with the PrimeScriptTM RT reagent kit (Takara, Diatech Lab Line, Ancona, Italy). RT-qPCR was performed with 25 ng of the template cDNA using the qPCRBIO SyGreen 2X master mix (PCR Biosystems, Resnova, Rome, Italy) and CFX384 (Biorad, Milan, Italy) was used as a detection system. YWHAZ and ACTB were used as housekeeping genes. Relative fold change was calculated using the ΔΔCt method [56 (link)] as described previously [57 (link)]. For primer design, the Primer-BLAST online tool [58 (link)] was used, and all primers (obtained both from Eurofins Genomics, Ebersberg, Germany, and Metabion International AG, Planegg/Steinkirchen, Germany) were tested for specificity and efficiency. Primer sequences are listed in Supplementary Table 1.

Total cellular RNA was isolated using an RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s instructions. The isolated total RNA was reverse-transcribed into cDNA using a High Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCR was performed using CFX384 (Bio-Rad, Hercules, CA, USA). mRNA expression levels were evaluated and normalized to β-actin levels as an endogenous reference. All primers for PCR were obtained from the TaqMan Gene Expression Assays (Thermo Fisher Scientific). The primers that were used are described in the Supplementary Methods.

Total mRNA was extracted from spleen and liver tissues with Trizol^® (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The purity and concentration of RNA samples were estimated by nucleic acid concentration analyzer NanoDrop 2000 (Thermo Fisher, Waltham, MA, USA) based on the ratio of the absorbance at 260 and 280 nm. The cDNA was synthesized from 1 µg of total RNA by reverse transcription in a 20 µL reaction using a PrimeScript^TM RT reagent Kit (Takara DRR037A, Dalian, China) following the manufacturer’s protocol. The expression levels of pertaining genes (β-actin, TNF-α, IFN-γ, IL-1β, IL-10, IL-6, Nrf2, HO-1, GPx1, NQO1, and GCLC) were analyzed by quantitative real-time PCR (CFX384, Bio-Rad, Hercules, CA, USA) using the SYBER^® Green PCR Master Mix (Applied Biosynthesis, Waltham, MA, USA), following the method according to our previous study [58 (link)] and the manufacturer’s instructions. The primer sequences used for each gene are presented in Table 2. The 2^-∆∆Ct method was used for quantification with the β-actin as a reference gene, and the relative abundance was normalized to the control (as 1) [59 (link),60 (link)]. The results were expressed as relative mRNA levels.