Rneasy mini kit

Total cellular RNA was isolated using RNeasy^® Mini Kit (Quiagen, Valencia, CA, USA). For tissue RNA isolation, the representative sample was collected from each carotid plaque. Frozen tissue was homogenized using a ball mill (Retsch, Haan, Germany), and mRNA was isolated using RNeasy^® Mini Kit (Quiagen). The total RNA amount was measured using a NanoDrop photometer (Thermo Scientific, Barrington, IL, USA). Reverse transcription was performed using GoScript™ reverse transcription system (Promega, Madison, WI, USA), according to the manufacturer’s instructions.

Islets were isolated by collagenase digestion as previously described (Rivera et al., 2014 (link)). Islets were manually picked, washed twice with ice-cold PBS, and frozen either in NP40 lysis buffer [20 mM Tris-HCl, 150 mM NaCl, 2 mM MgCl2, 0.5% NP-40, 1 mM DTT, 5 mM NaF, 1 mM Na3VO4, and protease inhibitor cocktail from Sigma (Cat#P2714, Sigma-Aldrich, St. Louis, MO, United States)] or in RLT buffer from Qiagen RNeasy MiniKit (Qiagen, Germantown, MD, United States) supplemented with bME (Sigma-Aldrich) at −80°C until use for protein and RNA analysis, respectively. To preserve a sample of pancreas for the immunohistochemical analysis, we ligated a small portion of the pancreatic tail with the surgical suture before perfusion to prevent the entrance of the perfusion solution, dissected it from the inflated pancreas, fixed with 4% PFA overnight, and embedded in paraffin.
Kidneys from the scid-beige mice transplanted with human islets were dissected before islet isolation from the pancreas. Left kidneys with transplanted islets were removed, the area containing islets was dissected, weighted, and homogenized in the ice-cold RLT buffer from Qiagen RNeasy MiniKit (Qiagen, Germantown, MD, United States) supplemented with bME (Sigma-Aldrich), and short-term stored frozen at −80°C before RNA extraction. Portions of the right kidneys were used as negative controls.

For RNA isolation, we cultured six populations of BM-MSCs between passages four and six on 60 mm dishes (BD Primaria™, BD Biosciences, San Jose, CA, USA) under standard growth conditions until a confluence of approximately 70% was reached. Then, incubation was started under experimental conditions: the first group was cultured in 2% O₂ (physical hypoxia), the second group was supplemented with 40 μM Vadadustat in 21% O₂ (pharmacological hypoxia), and the third was a control group growing under standard conditions (21% O₂). The incubation lasted for 6 h, after which we removed the medium from the cell cultures, washed cells with PBS and disrupted them by scraping in 350 μL of RLT isolation buffer from Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany). The cell lysates were then frozen at −80 °C for further use. We extracted total cellular RNA from BM-MSCs using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the protocol provided by the manufacturer. We determined the concentration and integrity of the isolated RNA samples using NanoDrop 1000 (NanoDrop Technologies, Thermo Fischer Scientific, Waltham, MA, USA) and Bioanalyzer Agilent RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA, USA).

500 μL of cell culture was centrifuged at 1000× g for 1 min at 4 °C; the resulting cell pellet was flash frozen by liquid nitrogen. Cells stored at −80 °C were resuspended with 600 μL buffer RLT 1% (v/v) 2-mercaptoethanol from the QIAGEN RNeasy Mini Kit (Cat No.: 74106), transferred to 2 mL OMNI prefilled ceramic bead tubes (SKU: 19-632), loaded onto an OMNI Bead Mill Homogenizer (SKU:19-040E) and agitated three times at 5 m/s for 1 min at 4 °C while cooled on ice for 3 min between each cycle. The resulting lysate was clarified by centrifugation at 11,000× g and used for total RNA extraction with QIAGEN RNeasy Mini Kit (Cat No.: 74106) with on-column DNase digestion. Extracted total RNA for each sample was evaluated for purity and quantified with the Qubit RNA HS Assay kit (Cat No.: Q32855) and an Invitrogen Qubit 2.0 Fluorometer (Cat No.: Q32866), each according to manufacturer’s instructions.
RNA libraries were prepared with Kapa stranded mRNA-seq kits, with KAPA mRNA Capture Beads (KAPA code: KK8421; Roche Cat No.: 07962207001) through the UNC High Throughput Sequencing Facility. All procedures were according to manufacturer’s instructions.

Total RNA was extracted from the serum samples using the Qiagen RNeasy Mini kit (Qiagen). Virus serotyping and genome copy numbers for each sample were determined using the DENV general-type real-time RT-PCR Liferiver kit (Shanghai ZJ Bio-Tech Co, China). Briefly, total RNA was extracted from 140μl of human serum using a Qiagen RNeasy Mini kit (Qiagen), as per manufacturer's protocol. RNA was eluted in 50μl of AVE buffer provided with the kit. 5μl of RNA was then added to the RT-PCR mix. The kits can detect 10³ copy/ml. Gene-specific primers were designed to amplify and sequence the envelope gene sequence from the virus. The D1-863F/2462R primer set (5’-TGCCATAGGAACATCCATCAC-3’ and 5’-TCCCAATGGCTGCTGATAGTC-3’) was used for RT-PCR amplification of the DENV-1 sequences and D2-556F/2160R (5’-GACCTTGGTGARTTGTGTGAAG-3’ and 5’-CARTCTTGTTACTGAGCGGA-3’) was used for RT-PCR amplification of the DENV-2 sequences. Both primer sets were also used for DNA sequencing of the viruses. The PrimeScript RT Reagent kit (TaKaRa) was used for reverse transcription and the PrimeSTAR GXL DNA Polymerase (TaKaRa) was used for PCR. Reverse transcription was conducted at 37°C for 15 min, 85°C for 5 s, followed by PCR amplification at 94°C for 2 min, 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min, with a final extension of 72°C for 5 min.

Total RNA was extracted using TRI-Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) coupled with Qiagen RNeasy Mini kit (Qiagen) following previously reported protocol (Lin et al., 2010 (link)). The potential DNA contaminant was eliminated using RQ1 DNase (Promega) according to the manufacturer’s protocol and the resultant RNA was purified using Qiagen RNeasy Mini kit. RNA concentrations were measured using NanoDrop ND-2000 Spectrophotometer (Thermo Scientific), and the qualities were assessed using the absorbance ratios of 260/280 nm and 260/230 nm.
For each sample, 300 ng total RNA was used in cDNA synthesis. GeneRacer oligo-dT (Invitrogen, Carlsbad, CA, USA) was used as the primer in the case where the resultant cDNA was for rhodopsin gene amplification. For cDNA to be used in RT-qPCR, oligo-(dT)₁₆ primer was used.