Nextseq 500 v2 kit

The genetic diversity analysis of I. cangae individuals was performed using the genome skimming approach (Figure 2a) (Malé et al., 2014 (link); Weitemier et al., 2014 (link); Wessinger et al., 2018 (link)). Shotgun paired‐end libraries were constructed from 50 ng of isolated DNA. For that, samples were subjected to a random enzymatic fragmentation in which the DNA was simultaneously fragmented and bound to adapters using the QXT SureSelect kit (Agilent Technologies). The fragmented DNA was purified using AmPure XP beads (Beckman Coulter) and subjected to an amplification reaction using primers complementary to the Illumina flowcell adapters. Amplified libraries were again purified using AmPure XP beads (Beckman Coulter), quantified using the Qubit 3.0 Fluorometer (Thermo Fisher Scientific Inc.), and checked for fragments size in the 4,200 TapeStation (Agilent Technologies®) using a ScreenTape DNA 1,000 kit (Agilent Technologies). The libraries were adjusted to a 4 nM concentration, pooled, denatured, and diluted to a running concentration of 1.8 pM. The sequencing run was performed in the NextSeq 500 Illumina platform using a NextSeq 500 v2 kit high output (300 cycles).

RNA was DNase I-treated, cleaned and concentrated (Zymo RNA Clean and Concentrator, Zymo Research) then enriched for poly(A) mRNA (NEBNext poly(A) mRNA Magnetic Isolation Module, NEB Biosystems, Ipswich, UK). Sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep kit (New England Biolabs). RNA and DNA quality were assessed using High Sensitivity RNA or DNA Screentape and an Agilent 4200 tapestation. Single-indexed and multiplexed samples were run on an Illumina Next Seq 500 sequencer using a NextSeq 500 v2 kit (FC-404–2005; Illumina, Can Diago, CA) for paired-end sequencing.
A computational pipeline was written calling scripts from the CGAT toolkit (https://github.com/cgat-developers/cgat-flow)^{39 (link),40 (link)}. Sequencing reads were de-multiplexed based on the sample index and aligned to the human genome assembly version 38 (GRCh38) using the STAR (Spliced Transcripts Alignment to a Reference) aligner^{41 (link)}. At least 14 million aligned reads were obtained per sample. Reads were mapped to genes using featureCounts v1.4.6 (part of the subreads package), in which only uniquely mapped reads were counted to genes. Differential expression analysis was performed using DESeq2^{42 (link)} between three groups: osteoclasts cultured on cell culture plastic, dentine or cellular cartilage.

RNA-seq of the MSC differentiation samples was performed according to the following protocol. 50,000 cells were seeded in 6-well plates in maintenance medium: MesenPRO-RS™ (Thermo Fisher Scientific, Massachusetts, USA). After 3 days, medium was replaced with osteogenic differentiation medium: DMEM high glucose supplemented with 10% FBS, 1× non-essential amino acids (NEAA), 2 mM L-glutamine, 0.28 mM ascorbic acid, 10 mM β glycerophosphate, and 10 nM dexamethasone (Sigma-Aldrich, USA). At each time-point: 0, 0.5, 1, 2, 4, 6, 8, 12, 16, 24, 48, 72, 125, 168, 336, and 504 h during osteogenesis, total RNA was isolated using TRIzol (Invitrogen™ Life Technologies, Carlsbad, USA) with the Direct-zol RNA kit (ZymoResearch, USA) according to the manufacturer’s instruction. RNA-seq library preparation was carried out using the NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs, USA) according to the manufacturer’s instruction. The quantity and quality of the cDNA library were assessed using the Agilent 2200 tapestation (Agilent Technologies, Santa Clara, USA). Paired-end sequencing of the pooled library was carried out using the Illumina NextSeq 500 v2 kit (Illumina, San Diego, USA) according to the manufacturer’s instruction.