Dp419

RNA extraction kits were used for obtaining total RNA (DP419, Tiangen, Beijing, China). The HiScript Q RT SuperMix for the quantitative real-time PCR (+gDNA wiper) Kit (R223, Vazyme, Nanjing, China) was used to produce first-strand cDNA from 500 ng of total RNA. ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme, Nanjing, China) and Light Cycler^® 480 II Real-Time PCR detection system (Roche, Basel, Switzerland) were used in the RT-qPCR. In this program, predenaturation at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 15 s and 72 °C for 30 s, and a final extension at 72 °C for 30 s. Table S2 listed primers used for RT-qPCR, as well as tomato Actin as an internal control. To calculate relative gene expression, the 2^−∆∆CT method was used, and a heat-map analysis was conducted using MEV version 4.9 (http://www.mev.tm4.org/ (accessed on 10 June 2020)). At the bottom, the intensity of the color bar showed the intensity of expression.

Cell cytoplasmic and nuclear extracts were prepared according to the described protocol (http://www.lifetechnologies.com/cn/zh/home/references/protocols/cell-and-tissue-analysis/elisa-protocol/elisa-sample-preparation-protocols/nuclear-extraction method-.html). RNA from the cytoplasmic extract was isolated according to the instruction of the kit (DP419, Tiangen Biotech, Beijing, China) and RNA from the nuclear pellet was extracted using TRizol (Thermo Fisher Scientific, San Jose, CA, USA). circ-231 was detected with real time RT-PCR and nuclear lncRNA MALAT1 were served as the nuclear control transcript 14 (link). Primers for PCR are shown in Supplementary Table S1.

Female flies were gathered 6–8 d post-eclosion for gene expression quantification and high-throughput sequencing. Fly heads were isolated by chilling them on liquid nitrogen and subsequent shaking. mRNA extraction was performed using Trizol according to a previously established protocol (Green and Sambrook, 2020 (link)). Genomic DNA was removed, and cDNA was synthesized using a commercial kit (TIANGEN#DP419). For real-time quantitative PCR, at least one PCR primer was designed to overlap with the sgRNA target site.
Genomic DNA from fly heads was extracted using a standard alkali lysis protocol (Huang et al., 2009 (link)). Genomic regions approximately 130–230 bp in length, centered around the sgRNA target site, were amplified by PCR employing Q5 polymerase (NEB#M0494). Subsequently, libraries were prepared using the BTseq kit (Beijing Tsingke Biotech Co., Ltd). These libraries were pooled and subjected to sequencing on the MiSeq platform (Illumina). Analysis of the libraries was conducted using Crispresso2 (Clement et al., 2019 (link)).

Total RNA was isolated using a total RNA extraction kit (DP419, TIANGEN) according to the manufacturer's instructions. One microgram of RNA was used as a template to synthesize cDNA using the GoScript Reverse Transcription System (PRA5000, Promega). All qPCRs were carried out using the Roche LightCycler System. The primers used in this study are from GenBank (https://pga.mgh.harvard.edu/primerbank/index.html) and are as follows: GAPDH, 5′-GTCTCCTCTGACTTCAACAGCG-3′ and 5′-ACCACCCTGTTGCTGTAGCCAA-3′; TRF2, 5′-GTGGAAAAGCCACCCAGAGAAC-3′ and 5′-TGCAAAGGCTGCCTCAGAATCC-3′; GPX4, 5′-ACAAGAACGGCTGCGTGGTGAA-3′ and 5′-GCCACACACTTGTGGAGCTAGA-3′; SLC7A11, 5′-TCCTGCTTTGGCTCCATGAACG-3′ and 5′-AGAGGAGTGTGCTTGCGGACAT-3′; p62, 5′-ACGCAGAACAGAGTTACGAAGGC-3′ and 5′-CCAGTCATCTTGTCCGTAGGCTTC-3′; and ATG5, 5′-GCAAGCCAAGGAGGAGAAGATTCC-3′ and 5′-GTGTCTCAGCGAAGCAGTGGTG-3′. Each sample was assayed in triplicate.
Relative mRNA levels were determined using the 2^−ΔΔCt method.

The methods used for RNA preparation and analysis were performed, as previously described (Yang et al., 2020 (link)). Briefly, total RNA of LV myocardial tissues were isolated using an RNA isolation kit (DP419, TIANGEN Biotech, China), according to the manufacturer’s protocol. Reverse transcription synthesis cDNA was amplified by SYBR Green SuperReal PreMix Plus (FP205, TIANGEN Biotech, China) by real-time fluorescent quantitative PCR amplification on an ABI 7500 PCR system (Applied Biosystems, United States). Primers were synthesized by Anhui General Biology Co., Ltd. and are listed in Table 1.

Three abdominal fat samples from each of HS or NS mice were examined. Total RNA was isolated using a commercial kit according to the manufacturer’s protocol (DP419, Tiangen, Beijing, China). After removal of genomic DNA, RNA was used to determine the digital gene expression profile (RiboBio, Guangzhou, China). Screening of differentially expressed genes (DEGs) was performed, and genes were considered to be DEGs with FDR <0.01 and a fold change ≥1.5. Gene Ontology enrichment analysis was performed using the GOEAST software toolkit. The significance level of GO term enrichment was set as FDR-adjusted p-value less than 0.05 by the Yekutieli method. Enriched KEGG pathways with DEGs were identified by a hypergeometric test using R packages (p < 0.01, FDR adjusted). Pathways with <3 known genes were discarded. Interactions between GO, KEGG, and other pathways were performed using IPA software.