Molecular grinding resin

Following cytokine treatment of monolayer HACs and MSCs, cell-to-cDNA lysates were treated with DNase and synthesized with cDNA using Moloney murine leukaemia virus (MMLV) reverse transcriptase (Life Technologies) according to the manufacturer’s instructions. MSC cartilage discs were harvested in TRIzol reagent (Life Technologies) and ground using disposable plastic pestles and Molecular Grinding Resin (G-Biosciences, St. Louis, MO, USA). Human articular cartilage was snap frozen, crushed and ground in liquid nitrogen using a pestle and mortar before RNA extraction using TRIzol reagent (Life Technologies). Total RNA was converted to cDNA using MMLV reverse transcriptase (Life Technologies) according to the manufacturer’s instructions.
Expression of genes of interest was measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR) on an Applied Biosystems ViiA 7 system using SYBR Green (Life Technologies). Relative quantification is expressed as the comparative cycle threshold (2^−ΔCt), where ΔC_t is C_t(gene of interest) − C_t(reference gene TBP or GAPDH). Samples where the reference gene C_t was greater than ±1.5 C_t from the median were excluded from further analyses.

For pellets that were cultured with TGFβ3 supplementation, we ground the pellets with Molecular Grinding Resin (G-Biosciences, Saint Louis, MO, United States, 786-138PR) extracted total RNA using Tri-Reagent (Sigma-Aldrich). Due to the considerably smaller size of the pellets cultured without TGFβ3 supplementation, we ground the pellets using the same grinding resin as above and used the RNeasy mini kit (Qiagen, Toronto, ON, Canada); 100 ng of the isolated total RNA was reverse transcribed into cDNA for qPCR analysis using the gene-specific primers listed in Supplementary Table 1. Expression of genes of interest was normalized to the mean expression level of reference genes YWHAZ, β-actin, and B2M (Foldager et al., 2009 (link); Munir et al., 2014 (link)) and presented using the 2ΔCt method (Schmittgen and Livak, 2008 (link)).

Genomic DNA was extracted from cells prior to the induction of chondrogenesis (Day0) and from Day14 cartilagenous discs by disruption in Invitrogen PureLink Genomic Digestion Buffer (Life Technologies, Paisley, UK) using a small disposable plastic pestle and an aliquot of Molecular Grinding Resin (G-Biosciences, St. Louis, MO) followed by proteinase K digestion for 1 hour at 37 °C then nucleic acid purification with Invitrogen PureLink Genomic DNA Kit according to manufacturer’s instructions (Life Technologies). 1 µg of genomic DNA was bisulphite converted using the EpiTect Bisulfite Kit (Qiagen, Manchester, UK). DNA methylation profiling of the samples was carried out by Cambridge Genomic Services (Cambridge, UK), using the Illumina Infinium HumanMethylation450 Beadchip array (Illumina Inc., San Diego, USA).