Ursus arctos
Protocols: Biopsies of the vastus lateralis muscle and skin tissue were collected from free-ranging brown bears (Ursus arctos), from Dalarna and Gävleborg counties, Sweden, in 2023. After collection, the samples were immediately frozen on dry ice until storage at -80°C. The same bears were captured during winter hibernation (in February) and during their active period (June). The study was approved by the Swedish Ethical Committee on Animal Experiment (applications Dnr C3/2016 and Dnr C18/2015), the Swedish Environmental Protection Agency (NV-0741-18), and the Swedish Board of Agriculture (Dnr 5.2.18–3060/17). All procedures complied with Swedish laws and regulations. Capture, anesthesia, and sampling were carried out according to an established biomedical protocol (Arnemo & Evans, 2017).  before RNA extraction, samples were first crushed in a Precellys lysing kit for hard tissue. Total RNA from bear skin were extracted with the NucleoMag RNA kit from Macherey-Nagel (744350.1). Tissues were lysed in 350µL of reconstituted Lysis Buffer (45-60% guanidium thiocyanate) and transferred in 1,5mL Eppendorf tube. RNA from samples have been extracted with the robot KingFisher Duo Prime Purification System. The following information describes the detailed procedure automatically performed by the robot. 350µL of binding buffer (95-100% 2-propanol) and 28µL of NucleoMag B-Beads were added to the samples. Samples were mixed by shaking for 5mn at RT. The supernatant was removed and samples were dried for 5mn at RT. DNA was digested in 300µL of rDNase reaction buffer. Samples were mixed and incubated 15mn at RT. 350µL of binding buffer was added and mixed by shaking for 5mn at RT. The supernatant was removed and samples were washed with 600µL of washing buffer 1 (15-30% guanidium thiocyanate, 35-55% ethanol). The supernatant was removed and samples washed twice with 900µL of washing buffer 2 (75-90% ethanol). Samples were air dried 15mn at RT and eluted in 50µL of elution buffer. Samples were heated at 70°C for 5mn before dosing.  -Take a certain amount of total RNA samples, and use oligo dT beads to enrich mRNA with poly A tail; -mRNA molecules were fragmented into small pieces; -The fragmented mRNA was synthesized into first strand cDNA using random primers; -The second strand cDNA was synthesized with dUTP instead of dTTP; -The synthesized cDNA was subjected to end-repair and 3' adenylated. Adaptors were ligated to the ends of these 3' adenylated cDNA fragments; -Digest the U-labeled second-strand template with Uracil-DNA-- Glycosylase (UDG) and perform PCR amplification; -Library quality control; -Library circularization; -The library was amplified to make DNA nanoball (DNB);  -Sequencing on DNBSEQ (DNBSEQ Technology) platform