Trizol reagent

Total RNA was isolated from the hippocampal regions of mice using TRIzol reagent (Omega Bio-Tek, Norcross, GA, USA). qRT-PCR of RARα, phosphatase, and tensin homologue deleted on chromosome 10 (PTEN), a disintegrin and metalloprotease 10 (ADAM10), calbindin 1 (Calb1), and erb-b2 receptor tyrosine kinase 4 (Erbb4) were performed by using the Mx3005P qPCR System (Stratagene, La Jolla, CA, USA). β-actin mRNA was chosen as the internal control gene, and the relative expression of mRNA was analyzed through 2^−ΔΔCT method, which was used for calculations.
All primers were commercially available from BGI Tech Solutions (Beijing, China). The primer sequences and primer ID are listed in Table 1.

Total RNA was extracted from sorted KCs, BMMϕs, and J774A.1 cells by TRIzol reagent (Omega Bio-Tek). cDNA was synthesized using the FastQuant RT Kit (TIANGEN). Real-time qPCR was performed as previously described (Xie et al., 2012 (link)). Target mRNA expression was normalized with β-actin and then was calculated using the 2^–ΔΔCT method. Primers were shown in Supplementary Table S1.

Total RNA was extracted from the spleen of Chinese giant salamanders using TRIzol Reagent (Omega Bio-tek, Norcross, GA, USA) and reversely transcribed into cDNA using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) following the manufacture’s protocol. Based on the transcriptome analysis of Chinese giant salamanders, a pair of primers AdMx-ORF-F (5′-ATGGGTAAAAAAAGGCCAAATC-3′)/AdMx-ORF-R (5′-GTTGTAGAACTTTTTAAGGGTCAA-3′) was designed to obtain ORF sequence. Two pairs of primers were designed according to the obtained ORF sequence: AdMx-3F outer primer (5′- GGTTGCTCAAACAAATGCAGAGTCTGAA-3′)/AdMx-3F inner primer (5′-GATACGTCTGAGGGAACGGATTGAACG-3′) and AdMx-5R inner primer (5′-CCACATTTCTTTAGTTCCTCTTTCGCTAC-3′)/AdMx-5R outer primer (5′-CATCAATGACATTCTTCTCTGTGCCTTTG-3′). Rapid amplification of cDNA ends (RACE) PCR was performed using the SMART RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) to identify the 5′ and 3′ untranslated region (UTR) of AdMx gene according to the manufacturer’s instructions separately. All the specific PCR products were purified on 1.5% agarose gels and then inserted into pMD19-T vector (TaKaRa, Taejin, Japan) for sequencing.

Total RNA was extracted using the TRIzol Reagent (OMEGA Bio-Tek). Reverse transcription was performed using the PrimeScript RT Reagent Kit (TaKaRa Biotechnology). All primers including PDGFRA, PDGFRB, FLT1, PDGFRL, and GAPDH were synthesized by Shanghai Sangon Co. (Sangon, Shanghai, China) Real-time PCR was performed using the SYBR Premix Ex Taq II Kit (TaKaRa Biotechnology).

The mixed plant tissues of FD and LYH during the annual growth season were used for full-length transcriptome analysis. Total RNA was extracted with TRIzol reagent (Omega BioTek, GA, USA). After qualified samples were detected, full-length cDNA was synthesized, followed by PCR amplification, terminal repair, splicing, exonuclease digestion, and a sequencing library was obtained. After the library was validated, full-length transcriptome sequencing was performed using PacBio RS II platform. After a series of processing, the full-length transcripts were functionally annotated [92 (link)].