GSM6208927: CTRL 4; Mus musculus; RNA-Seq
Bones were cleaned of soft tissue, and the distal and proximal epiphyses were excised. Bone marrow was removed by centrifugation at >20 000 g for 15 seconds at 4 °C. TRIsure was added to marrow-free bones, which were then flash frozen in liquid nitrogen and stored in -80 °C. For RNA isolation, both tibiae and femurs from each mouse were subjected to 1 cycle of 45 seconds at full speed using a Polytron (Kinematic PT). Total RNA was separated from bone minerals, DNA and protein by phenol-chloroform extraction. The RNA-containing fraction was then purified using the RNeasy Mini Kit (Qiagen) and treated with DNase I (RNase-Free DNase Set, Qiagen). DNA-free RNA samples were quantified with the Qubit® RNA BR Assay kit (Thermo Fisher Scientific), and RNA integrity was estimated with the Agilent RNA 6000 Pico Bioanalyzer 2100 Assay (Agilent). The RNASeq libraries were prepared with the KAPA Stranded mRNA-Seq Illumina® Platforms Kit (Roche) following the manufacturer´s recommendations. Briefly, 500 ng of total RNA was used for enrichment of the poly-A fraction with oligo-dT magnetic beads, following mRNA fragmentation. Strand specificity was achieved during the second-strand synthesis performed in the presence of dUTP instead of dTTP. The blunt-ended double stranded cDNA was 3´adenylated, and Illumina platform-compatible adaptors with unique dual indexes and unique molecular identifiers (Integrated DNA Technologies) were ligated. The ligation product was enriched with 15 PCR cycles. The size and quality of the libraries were assessed in a High-Sensitivity DNA Bioanalyzer assay (Agilent).