Pcd2.1 vector

The second exon sequence of the INSR gene was inserted into the pCD2.1 vector (Geneseed Biotech, Guangzhou, China) and psi-CHECK2 vector (Promega, Fitchburg, WI, USA). Small interfering RNA (siRNA) oligonucleotides were designed to combine with the back-splice region of circINSR (RiboBio, Guangzhou, China). These siRNAs inhibited the expression of circINSR after transfection into the cell and was named si-circINSR. The mimics of bta-miR-15a, bta-miR-15b, bta-miR-16a, and bta-miR-16b were purchased from RiboBio (Guangzhou, China). The 3′-untranslated regions (UTRs) of the cyclin D1 (CCND1) and B-cell lymphoma 2 (Bcl-2) genes containing the miR-15/16 binding sites were amplified using the PCR enzyme mix (Platinum II Taq Hot-Start DNA Polymerase, Invitrogen). The wild-type and mutant 3'-UTR gene sequences were cloned into the psi-CHECK2 vector. The Renilla: Firefly ratio was measured and compared against that for the non-treated control. The mimics (50 nM) or vectors (2 μg/mL) were transfected into cells using a transfection reagent (R0531, Thermo Fisher Scientific, USA). For the overexpression of the miR-15/16 family, the miR-15a, miR-15b, miR-16a, and miR-16b mimics were mixed in equal amounts for transfection.

The full‐length cattle and mouse circNEB sequences were cloned by back‐to‐back primers, respectively, and cloning primers are listed in Table S3 (Supporting Information). The cloned fragments were linked to the pCD2.1 vector (Geneseed, Guangzhou, China) through Kpn I and BamH I restriction enzyme sites. The pCD2.1‐bos‐circNEB and pCD2.1‐mus‐circNEB vectors were obtained. pCD2.1‐bos‐circNEB vector was used to overexpress circNEB in fetal cattle myoblasts. pCD2.1‐mus‐circNEB vector overexpressed circNEB in C2C12 cells. Cas9 knockdown vector was constructed by applying px458 plasmid and ligated by restriction enzyme cleavage site Bbs1.^[32 (link)
^] The knockdown site was located within 1 kb bases of the intron on both sides of circNEB, and the corresponding sgRNA sequences have been listed in Table S3 (Supporting Information). The overexpression plasmid was transfected into the cells by Exfect Transfection Reagent (Vazyme, Nanjing, China). The transfection method was performed in reference to the instruction manual.