Dose-dependent progression of multiple low dose streptozotocin-induced diabetes in mice - DEGs

Differentially expressed genes in mice treated with multiple low doses of streptozotocin.

        All animals were maintained in accordance with City of Hope Institutional Animal Care and Use Committee protocols (#16047) in accordance with the Guide for the Care and Use of Laboratory Animals (11). Only male C57BL/6J were used due to the varying effectiveness of STZ in female mice. All mice were housed in individually ventilated cages with 3-5 mice/cage. Animals were maintained on 12-h light:dark cycles with ad libitum access to water and standard rodent chow diet.

        STZ (Sigma-Aldrich S0130-1G; St. Louis, MO, USA) was administered for five consecutive days by intraperitoneal (IP) injections of STZ dissolved right before the injection in saline at either 35 or 55 mg/kg body weight. Saline was administered to 34 mice as a vehicle control, 40 mice received 35 mg/kg STZ, and 38 mice received 55 mg/kg STZ. Equivalent volumes of saline were used in control animals. Approximately 75-100 healthy islets per mouse were picked by hand, washed in cold PBS, and then resuspended in 350 ul of RLT with 1%BME. RNA was isolated using RNeasy micro (Qiagen 7404; Hilden, Germany) kits according to manufacture instructions and DNA was digested by treatment with RNAse-Free DNAse Set (Qiagen 79254). 

       RNA sequencing libraries were prepared using 150 ng total RNA with Kapa RNA mRNA HyperPrep kit (Kapa Biosystems, Cat KR1352; Cape Town, South Africa) to enrich for mRNA according to the manufacturer's protocol with a final PCR cycle of 12x. The RNA fragment time was 8 min at 94 C; 1.0x beads purification was used for the final sequencing library purification. The final libraries were validated with the Agilent Bioanalyzer DNA High Sensitivity Kit and quantified with Qubit.  Sequencing was performed on Illumina NovaSeq 6000 by using S4 kit v1.5 with the paired end mode of 2x101cycle. Real-time analysis (RTA) v3.4.4 software was used for base calling. The number of biological replicates were as follows; saline control: 6, STZ, 35 mg/kg: 7, STZ, 55 mg/kg: 8.