Pgem t

The fertilized eggs, 2-cell embryos, high blastulae and gastrulae, 10-somite larvae, and 1-, 2-, and 3-day-old larvae were collected. A fragment of elavl1a was PCR-amplified using the primer pair P7 and P8 (Supplementary Data 1) and subcloned into vector pGEM-T (Tiangen Biotech, Beijing, China). Digoxigenin (DIG)-labeled elavl1a-specific antisense riboprobes were synthesized with linearized vectors (digested by Nco I enzyme) and Sp6 RNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) through in vitro transcription. Whole-mount in situ hybridization was performed by the method of Thisse and Thisse^{33 (link)}. After staining, the embryos/larvae were observed and photographed under Nikon SMZ1000 stereomicroscope.

Real-time PCR was carried out on an IQ5 detection system (Bio-Rad, San Diego, CA, United States), using SYBR green mixture reagent (Qiagen, Dusseldorf, Germany) in a volume containing 12.5 μL SYBR green mixture, 100 nM of each primer, and 1.0 μL template DNA. The primer pairs of uniMet1-F and uniMet1-R were used to amplify the partial methanogen 16S rRNA gene (Zhou et al., 2010 (link)), with the following amplification conditions: denaturation at 95°C for 10 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s. The primer pairs of Bac1 and Bac2 were used to amplify the partial bacterial 16S rRNA gene using the same conditions as above (Chin et al., 2010 (link)). The vector pGEM-T (Tiangen, Beijing, China) with inserted bacterial and archaeal fragments was used to amplify standard curves and determine copy numbers.