title: GSM3902353: A_minus_509.2_tube.2.509.2.L.tibia; Mus musculus; RNA-Seq
library_construction_protocol: Four hours after the last loading bout, right and left tibiae were stripped of all muscle tissue and flushed by centrifugation (10,000g x 2 min) at room temperature to remove bone marrow.  The flushed tibiae were then cut 2 mm from the articular surface and again at the distal TFJ to isolate the area of peak strain.  Tissue samples were flash-frozen in liquid nitrogen and stored at -80C°.  Frozen tibiae were pulverized in liquid nitrogen using a Mikro Dismembrator (Mikro-Dismembrator S; B. Braun Biotech International, Melsungen, Germany), resuspended in TRIzol (15596026; Invitrogen, USA), and stored at -80C°.  Total RNA was isolated using a Direct-zol kit per manufacturer instructions (R2072; Zymo Research, USA).  RNA concentration was determined using a Nanodrop (ND-2000; ThermoFisher, USA).  RIN values were used to determine RNA quality (Bioanalyzer 2100; Agilent Technologies, USA), and the average RIN score was 5.4. RNA sequencing was performed on the Total RNAble platform (Cofactor Genomics, http://cofactorgenomics.com, St. Louis, MO, USA).  Briefly, species-specific rRNA-probes were hybridized to total RNA to remove nuclear-encoded and mitochondrial contaminating ribosomal RNA from the sample.  The resulting ribo-depleted RNA was then fragmented.  First-strand cDNA synthesis was performed using reverse transcriptase and random primers in the presence of Actinomycin D, followed by second-strand cDNA synthesis with DNA polymerase I and RNase H.  Double-stranded cDNA was end-repaired and A-tailed for subsequent adaptor ligation.  Indexed adaptors were ligated to the A-tailed c-DNA.  Enrichment by PCR was performed to generate the final cDNA sequencing library.  Libraries were sequenced as single-end 75 base pair reads on an Illumina NextSeq500 following the manufacturer's protocols.