Truseq v2 library preparation kit

Total RNA from flushed and minced femoral bones or cell cultures was isolated using the Direct-Zol RNA Mini Prep Kit (Zymo Research) following the supplier’s instructions. For quantitative real-time polymerase chain reaction (rt-qPCR), cDNA was synthesized from ∼0.5 μg RNA using the SuperScript III first strand synthesis system (Invitrogen) as described by the supplier and subjected to rt-qPCR amplification using the QuantiTect SYBR-Green PCR Kit (Qiagen) and a CFX384 real time system machine (Bio-Rad) using the following conditions: 10 min 95 °C followed by 45 cycles of 30 sec at 95 °C, 30 sec at 60 °C and 30 s at 72 °C. Primers for the analyzed genes are listed in Table S1. All rt-qPCR assays were performed in triplicate and expression was evaluated using the Biorad CFX Manager 3.0 software applying the comparative quantification method^{75 (link)}. For high through output next generation RNA sequencing (RNA-seq) RNA integrity was analyzed with a 2100 Bioanalyzer with an RNA 6000 kit (Agilent Technologies). RNA-Seq libraries were prepared using the Illumina TruSeq v2 library preparation kit and sequenced with paired-end 50 bp reads on an Illumina HiSeq 4000 in biological triplicates for bone tissues (femurs) and duplicates for cell-lines.

Total RNA was derived from the whole blood, depleted of globin transcripts using Ambion GLOBINclear, and subsequently processed using the Illumina TruSeq v2 library preparation kit. On average, 40 million paired-end reads of 50 bp were generated per participant using illumina’s Hiseq 2000. Samples were demultiplexed using CASAVA and aligned to the hg19 reference genome using STAR [43 (link)]. Alignments were sorted, read groups were added using picard [44 ], and gene expression was quantified using featureCounts [45 (link)]. We selected participants for which all covariates were available (sex, age, BMI, smoking status, and measured cell counts). Raw count matrices per cohort were used for analysis.