Rna seq

Red panda eMSCs isolated from three different individuals and corresponding red panda skin fibroblast cells (skin FCs) at passage 4 were seeded in culture dishes (diameter, 10 cm) and treated with normal growth medium. When the cells reached ~80% confluence, the cells were collected and treated with Trizol (Thermo Fisher) as manufacturer’s protocol to extract total RNA and then for RNA-seq (Novogene). The RNA-seq data were assembled and analysed as no reference genome sequences. Differential expression analysis was performed with DESeq2. For functional enrichment analysis, DEGs were mapped to terms in the GO database, and then searched for significantly enriched GO terms (P < 0.05). DEGs were mapped to the KEGG database, and searched for significantly enriched KEGG pathways (P < 0.05).

Construction of the cDNA libraries and RNA-Seq were performed by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). First, mRNA was purified from 1.5 μg of total RNA from salivary gland tissue using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperatures in NEB Next First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Subsequently, second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After the adenylation of 3’ ends of DNA fragments, NEBNext Adaptors with a hairpin loop structure were ligated to prepare for hybridization. The library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA) to select cDNA fragments that were preferentially 150~200 bp in length. Then, 3 μL of USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then, PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, PCR products were purified (AMPure XP system), and library quality was assessed on an Agilent Bioanalyzer 2100 system.

Total RNA from hCGp7 cells used for qPCR was subjected to RNA-seq (Novogene, Beijing, China). RNA qualities were checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). RNA integrity number values were 10.0 in all samples. The RNA was subjected to prepare libraries using Illumina TruSeq RNA and DNA Sample Prep Kits (Illumina, San Diego, CA, USA). Library qualities were confirmed using a Qubit 2.0 fluorometer (Life Technologies; Thermo Fisher Scientific) and an Agilent 2100 Bioanalyzer. RNA-seq was performed using Illumina NovaSeq 6000 (Illumina) to generate >6 GB raw data per sample. Raw data were recorded in a FASTQ format. The quality of the read was calculated as the arithmetic mean of its Phred quality scores. Then the reads as follows were discarded with adaptor contamination, when uncertain nucleotides constitute >10% of either read, or when low quality nucleotides (base quality < 20) constitute >50% of the read. The cleaned reads were used for subsequent analyses. The reads were mapped to a reference genome GRCm39 using TopHat2. The numbers of the reads and mapping efficiencies were summarized in Table 1. The expression levels of the transcripts were calculated as FPKM using HTSeq.