Abi 3700 sequencer

WES was performed in all three patients as previously described (Wang et al., 2014 (link); Wang et al., 2016 (link)). Briefly, genomic DNA was extracted from patients’ peripheral blood and was then sheared to create fragments of 150 to 200 bp. Sequencing library was prepared using the SureSelect XT Human All Exon V6 kit (Agilent Technologies, Santa Clara, CA, United States), and sequencing was performed by the Illumina NovaSeq 6000 System (Illumina, San Diego, CA, United States). After base calling, quality assessment, and alignment of the sequence reads to the reference human genome (GRCh37, dbSNP135), all single nucleotide variants (SNVs) and indels were saved as VCF format file which was then uploaded to the QIAGEN Clinical Insight (QCI) Interpret Translational tool (https://apps.qiagenbioinformatics.cn/) for filtering and annotation. The SOX11 variants identified by WES were validated by Sanger sequencing using the ABI 3700 sequencer (Applied Biosystems, Foster City, CA, United States), in indicated patient and their parents (sequencing primers were available upon request).

To determine the status of methylation of MALAT1 and miR-146a promoters in each collected sample, DNA extracted from the samples was modified first with bisulfite (Zymo Research, Orange, CA, United States) based on the protocol provided by the reagent manufacturer and then subjected to PCR sequencing on an ABI 3700 sequencer (Applied Biosystems, Waltham, MA, United States) based on the protocol provided by the equipment manufacturer.

Adult seahorses were anesthetized with 0.05% MS222 (Sigma, Shanghai, China). Total RNA was extracted from the brain using TRIzol reagent (Invitrogen, CA, United States) according to the manufacturer’s instructions. First-strand cDNA was synthesized using the PrimeScript^TM RT Reagent Kit with gDNA Eraser (Takara, Dalian, China). Partial cDNA fragments were obtained by PCR using primers (Table 1) designed based on the sequences of kiss2 and GPR54-2 from the published genome data of lined seahorse (Lin et al., 2017 (link)). To obtain the full-length sequence of the cDNA of the kisspeptin gene in lined seahorse, 3′ and 5′ rapid amplification of cDNA ends (RACE) PCRs were conducted using the SMARTer RACE Kit (Clontech, CA, United States) according to the manufacturer’s protocols. PCR amplification was performed using Taq DNA Polymerase Mix (TAKARA, Dalian, China), and PCR conditions were as follows: denaturation at 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 56°C for 30 s, 72°C for 2 min, and a final incubation at 72°C for 10 min. The PCR products were purified from an agarose gel and subcloned into a pGEM-T Easy Vector System (Promega, Fitchburg, WI, United States). The inserts were sequenced using an ABI 3700 sequencer (Applied Biosystems).

Bacterial DNA was extracted following the method described by Marmur [19] . The 16S rRNA gene was amplified by PCR using the conserved primers 27F [20] (link) and 1522R [21] (link) with the following PCR thermal conditions: 95°C for 1 min; 35 cycles of 95°C for 15 s, 55°C for 15 s, 72°C for 2 min; and a final extension cycle at 72°C for 10 min. Forward and reverse strands of the amplified DNA fragment were sequenced in an ABI 3700 sequencer (Applied Biosystems). The identification of phylogenetic neighbours was carried out by the BLAST program [22] (link) against the NCBI database and the database of type strains EZtaxon [23] with validly published prokaryotic names.