title: RNA Seq of Jellyfish: NFMP
design: Total DNA was extracted using a DNeasy PowerSoil Pro Kit (QIAGEN,                 Germany) and tested using 1% agarose gel electrophoresis. PCR amplification for the                 16S rRNA gene (V4 region) was conducted with primers 515F (5-GTGCCAGCMGCCGCGGTAA-3)                 and 806R (5-GGACTACHVGGGTWTCTAAT-3) (Hugerth et al., 2014) using an ABI GeneAmp 9700                 thermocycler (USA). The PCR cycle included initial denaturation at 95 C for 3 min;                 29 cycles of 95 C for 30 s, annealing at 53 C for 30 s, and 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                 FastPfu buffer, 2 L of 2.5 mM dNTPs, 0.8 L of each primer (5 M), 0.4 L FastPfu DNA                 polymerase (TransGen Biotech, China), 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). Library preparation was performed using a TruSeq DNA                 Sample Prep Kit (Illumina, USA), and sequencing was conducted on an Illumina MiSeq                 platform following the protocols of Majorbio Bio-Pharm Technology Co., Ltd.                 (Shanghai, China).