Primestar gxl dna polymerase

The complete sequence of linc‐ROR was amplified by using a high‐fidelity enzyme (MCLAB, San Francisco, CA, USA) to perform PCR, and the pmirGLO Dual‐luciferase miRNA Target Expression Vector (Promega, Madison, WI, USA) was digested by the Sac I (NEB, Ipswich, MA, USA) and XhoI (NEB) enzymes. Then, these two parts were ligated into a recombinant plasmid by the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). The recombinant linc‐ROR‐WT plasmid was verified by sequencing. The predicted binding sites between linc‐ROR and miR‐194‐3p were mutated by PCR (PrimeSTAR GXL DNA Polymerase; Takara, Kusatsu, Shiga, Japan) to construct the linc‐ROR‐MUT plasmid. Likewise, the 3′‐UTR of MECP2 was amplified by PCR (PrimeSTAR GXL DNA Polymerase; Takara), and then, MECP2‐WT and MECP2‐MUT were constructed as mentioned above. The primers used are shown in Table 2.
Then, 4 μg of Linc‐ROR‐WT/MUT or MECP2‐WT/MUT and 100 nm miR‐194‐3p mimic or NC mimic (RiboBio) were cotransfected into HEK‐293T cells. After culturing for 48 h, the cells were fully lysed and collected to detect Firefly luciferase value and Renilla luciferase value by the Dual‐Luciferase Reporter Gene Assay Kit (Beyotime, Shanghai, China) under the Promega GloMax 20/20 Luminescence detector. The RLU ratio (firefly fluorescence value/Renilla fluorescence value) was calculated.

The library construction and PacBio sequencing were performed according to the official protocol as described by Pacific Biosciences (Pacific Biosciences, USA). Briefly, 1 μg of total RNA was used as input for first-strand cDNA synthesis using a SMARTer PCR cDNA Synthesis kit (Clontech, USA). The first-strand products were diluted to an appropriate volume and subsequently used for large-scale PCR. Next, a total of 12 PCR cycles of amplification were performed for second-strand cDNA synthesis using PrimeSTAR GXL DNA Polymerase (Clontech, USA). After amplification, the PCR products were purified with AMPure PB Beads (Pacific Biosciences) and then normalized by repairing DNA damage, repairing ends and blunt ligation reactions. The normalized cDNA products were then subjected to the construction of SMRTbell template libraries using the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences). Finally, two SMRT cells were sequenced on a PacBio Sequel instrument using sequencing kit 2.1 (Pacific Biosciences) with 10 h movie recordings.

Total DNA was extracted from 10–20 male flies using the DNeasy Blood and Tissue kit (Qiagen) and following manufacturer’s instructions. PCR amplification was performed using PrimeSTAR GXL DNA Polymerase under manufacturer’s conditions (Clontech, RRID:SCR_004423), with 1 μM of primers and the following amplification conditions: 94 °C, 1 min; 98 °C, 30 s; 68 °C, 13 min (30 cycles); 72 °C, 10 min. Primers used (14.2F: 5′-GCCGCTCCTTTCCATTTTTGATTTCC and 14.2R: 5′-TGCCAGCAGTCGCGGTTATACCA) amplify a product encompassing almost the complete mtDNA molecule. The PCR products were then visualized after electrophoresis on 0.8% agarose and 2X Invitrogen SYBR Safe DNA Gel Stain (Thermo Fisher Scientific, RRID:SCR_008452) and 1 kb DNA Ladder (New England Biolabs, RRID:SCR_013517).