Pcdna3.1 vector

The full length of Circ‐9110 was extended by PCR and then cloned in a pcD2.1‐cir vector (Geneseed Biotech, Guangzhou, China). Given that the CDSs of HOXA1 X1 were not amplified in dairy goats, the CDSs of HOXA1 X2 were cloned and inserted in pcDNA3.1(+) vectors (Promega, Madison, USA).
MiR‐100‐5p mimic, NC mimic, miR‐100‐5p inhibitor, NC inhibitor, Circ‐9110‐siRNA and HOXA1‐siRNA were synthesized with Ribobio (Guangzhou, China). The ESCs were seeded at a density of 7.5 × 10⁵ cells/well in 6‐well plates and then transfected at 60% confluency with MiR‐100‐5p mimic, miR‐100‐5p inhibitor, NC mimic or NC inhibitor, pcDNA3.1, pcDNA3.1‐HOXA1 and pcD2.1‐ciR or pcD2.1‐circ‐9110 at 100 nM final concentrations by using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's specifications.

Genes encoding human IgG1 and mouse IgG2a heavy chain Fc fragments were inserted, respectively, into pcDNA3.1 vector (Life Technologies, Carlsbad, CA, USA) at its multiple cloning sites to construct different Fc-fusion protein expression vectors. The published sequences of human IgG1-Fc and mouse IgG2a-Fc were acquired [32] (link)–[33] (link). The gene encoding human IgG1 (hinge-CH2-CH3) was obtained by PCR using the following primers: sense 5′-CGCAAGCTTTAAGGATCCATCTGCAACGTGAATCAC-3′ and antisense 5′-TGCGAATTCTTACGTCGCACTCATTTACCCGGAGAC -3. The amplified DNA fragment in a sequence order of Hind III+BamHI+ human IgG1 (hinge-CH2-CH3)+ XbaI was first cloned into T-easy vector (Promega Corporation, Madison,WI, USA) and subcloned into pcDNA3.1 vector by HindIII and XbaI digestion. This vector, pCDH, was used to express CRDs of the CD137 extracellular region with its own leader sequence. DNA sequence of “HindIII+HE4 leader+AgeI+human IgG1(hinge-CH2-CH3)+XbaI” and “HindIII+HE4 leader+AgeI+mouse IgG2a(hinge-CH2-CH3)+XbaI” were synthesized by Sangon Biotech (Shanghai, China) and then subcloned into pcDNA3.1 vector to construct two other expression vectors, pCDH-L and pCDM-L, both containing a HE-4 (human epididymis protein 4) leader sequence. These two vectors were used for expressing the genes of CD137L extracellular region and truncated CD137 CRDs.

The human gene encoding OR10A3 was purchased from Novoprolabs (Cat# 746761-1; Shanghai, China) and subcloned into the pcDNA3.1 vector (Promega, Madison, WI, USA). Then, HEK293T cells were seeded into a 24-well plate (100,000 cells/well) and cultured for 24 h. The recombinant plasmid containing the OR10A3 was subsequently co-transfected into the HEK293 cells with (1) Renilla luciferase reporter plasmid (pRL-TK; Promega, Madison, WI, USA), (2) cAMP response element firefly luciferase construct (pCRE-luc; Promega), and (3) accessory proteins that facilitate the translocation of the olfactory receptor to the cell membrane (RTP1S, Ric8B, and Golf; gifts from Hiroaki Matsunami at the University of Duke in the USA and Cheil Moon at Daegu Gyeongbuk Institute of Science and Technology (DGIST) in Korea), using lipofectamine 3000 transfection reagent (Thermo Fisher Scientific). 48 h after transfection, cells were treated with vehicle control or suberic acid (100 uM), incubated for further 6 h, and harvested for dual-luciferase assay using a Dual-Glo Luciferase assay system (Promega). The firefly luciferase signal was measured first and followed by Renilla luciferase in the same sample using a GloMax 20/20 luminometer (Promega).

pcDNA-3.1-c-JUN expression vector. The c-JUN coding sequence was amplified from chicken leg muscle cDNA using PCR and the following specific primers: 5′-CCGCTCGAGGCCACCATGGAGCCTACTTTCTACGAG-3′ and 5′-CGGGGCCCCGCTTCTACCGTCAGCTTTAC-3′. The PCR product was cloned into the pcDNA-3.1 vector (Promega, Madison, WI, USA) using the XhoI and ApaI restriction sites.

Amplify the full-length CDS sequence of DYNLL2 from chicken breast muscle cDNA, and use Hind III and EcoR I restriction enzyme sites to construct an overexpression plasmid using pcDNA3.1 vector (Promega, Madison, WI, USA). For the construction of the psiCHECK^TM-2 dual-luciferase reporter vector (Promega, Wisconsin, USA), we first predicted the binding sites between miR-148a-3p and DYNLL2 on the Targetscan website (https://www.targetscan.org/) [49 ], the wild-type sequences of the DYNLL2 3’UTR that contained the putative gga-miR-148a-3p binding sequence TGCACTG were amplified by PCR using chicken breast muscle cDNA, then use Xho I and Not I restriction enzyme sites to construct the wild-type sequence on the psiCHECK ^TM-2 vector. The mutant-type sequences of the DYNLL2 3’UTR were generated by changing the binding site of gga-miR-148a-3p from TGCACTG to ACGTGAC. The primer sequences are listed in Table S5. To investigate the binding sites of the DYNLL2 3’UTR with miR-148a-3p, DF-1 cells were cotransfected with the DYNLL2-WT or the DYNLL2-MUT reporter plasmids individually, in combination with the mimics NC or miR-148a-3p mimics, respectively. At 48 h posttransfection, firefly and renilla luciferase activities were measured by a Dual-Luciferase Reporter Assay System (Promega, USA) following the manufacturer’s instructions.