Turbo dnase 1

Specimens were collected from Wenshan (Yunnan) and Wangmo (Guizhou) between 2019 and 2023. All individuals were maintained on moist soil in the laboratory before RNA extraction and chromosome preparation (animal handling and experiments were approved by the Ethics Committee at Guizhou Normal University, Permission number: 20,230,300,015). The experiments adhered to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines [36 (link)]. All methods were conducted in compliance with relevant guidelines and regulations.
The specimens were euthanized using ethyl acetate and then preserved in 70% alcohol. For maximum mRNA extraction, five representative organs (brain, heart, liver, skin, and muscle) were collected and mixed from two healthy adult females. Total RNA was extracted from the mixed tissues using TRIzol reagent (Invitrogen, MA, USA) on dry ice, following the manufacturer’s instructions. DNA was removed using TURBO DNase I (Promega, Beijing, China). RNA degradation and contamination were assessed by 1% agarose gel electrophoresis. RNA purity was determined using a NanoDrop 2000 microspectrophotometer (Thermo Scientific, USA; NanoDrop 2000 detection blank reference: DEPC water). The RNA integrity (RIN) was accurately measured with an Agilent 4200 (Agilent Technologies). Only RNA samples with a RIN ≥ 8 were considered suitable for cDNA library construction.

Tissue specimens corresponding to the various brain regions (cerebellum, brain stem, cerebral cortex and basal ganglia) were dissected from the brain of AS and AS/AGU rat strains, snap-frozen in liquid nitrogen, and stored for further analysis. Total RNA was isolated using TRI^®zol reagent according to the manufacturer’s instructions, followed by DNase treatment to remove genomic DNA contamination (as per the manufacturer’s instructions, Turbo DNase I, Promega, Southampton, UK). RNA quality and quantity was estimated spectrophotometrically (NanoDrop^®2000, Thermo Fisher Wilmington, USA) and 500 ng of total RNA was subjected to reverse transcription using SuperScript II^® (Invitrogen, Paisley, UK). QPCR was performed using pre-designed TaqMan^® probes: Cdkn2A/p16 (Rn00580664_m1), Sirt5 (Rn01450559_ml), Sirt6 (Rn01408249_ml), Sirt7 (Rn01471420_ml), and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Rn01775763_g1) as an endogenous control according to the manufacturer’s protocols. Each sample was run in triplicate and a standard deviation between cycle threshold (Ct) values of less than 0.25 was considered as acceptable for further analyses. Relative gene expression was calculated using the comparative threshold (Ct) method [30 (link)].

Xiaoyan 81 was cultivated in the field as described previously [35 (link)]. After heading, the plants were inspected regularly to record the timing of anthesis and grain development. Unfertilized caryopses were collected from 10 different main stem spikes approximately 2 days before anthesis. The grain samples were similarly collected at 5, 15 and 25 DAA, respectively. Total RNA samples were isolated from unfertilized caryopses and developing grains using a commercial Kit (Takara Biotechnology, Dalian, China). The purified RNA was dissolved in RNase-free water, with genomic DNA contamination removed using TURBO DNase I (Promega, Beijing, China). The integrity of the RNA thus prepared was determined with the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, California). Only the total RNA samples with RIN value ≥ 8 were used for constructing the cDNA libraries in PacBio or HiSeq sequencing.