RNA Seq of Jellyfish: Pd
Total DNA was extracted using a DNeasy PowerSoil Pro Kit (QIAGEN, Germany) and tested using 1% agarose gel electrophoresis. PCR amplification for the full-length 16S rRNA gene (FL-16S) was conducted using primers 27F (5-AGRGTTYGATYMTGGCTCAG-3) and 1492R (5-RGYTACCTTGTTACGACTT-3) (Nilsson et al., 2019) with an ABI GeneAmp 9700 thermocycler (USA). The PCR cycle included initial denaturation at 95 C for 3 min, followed by 29 cycles of 95 C for 30 s, annealing at 60 C for 30 s, extension at 72 C for 45 s, and a final extension at 72 C for 10 min. The PCR reaction mix contained 4 L of 5  KAPA HiFi buffer, 0.4 L KAPA HiFi DNA polymerase (Kapa Biosystems, USA), 0.8 L of each primer (5 M), and 10 ng of template DNA in a 20 L volume. The PCR products were electrophoresed on a 2% agarose gel, purified with an AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, USA), and quantified using QuantiFluor-ST (Promega, USA). FL-16S amplicons were prepared for sequencing using the PacBio SMRTbell adapter ligation and DNA damage repair protocol. Sequencing was performed on a PacBio SMRTLink (v11.0) platform following the protocols of Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).