Transcriptomic Analysis of Osteoclast Differentiation

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.