GSM4594471: Rat_tibia_R3_TAGCTT; Rattus norvegicus; RNA-Seq
To obtain highly enriched populations of osteocytes, tibias of rat were cut to expose the metaphysis and centrifuged briefly to remove bone marrow (1500g, 30 sec).  Calvarial samples were obtained from the rodents with scissors, removing the sutures and avoiding any marrow spaces (diploe). All samples were dissected free of periosteum and accessible surfaces were scraped with a scalpel. They were then immersed briefly in a 1mg/ml solution of collagenase (Sigma Aldrich) for 3-5 minutes at 37°C to remove any adherent surface cells.  Samples were then washed in saline before being snap frozen by complete submersion in liquid nitrogen and then stored at -80˚C.  Bone samples were pulverised using a mikro-dismembrator-S (Braun Biotech International GmbH Melsungen, Germany), in which the bone is shaken in a robust PTFE mill chamber with an 8mm tungsten carbide ball (both cooled in liquid nitrogen before use, and again after adding the bone pieces, before agitation).  The mill with tissue sample was placed within the mikro-dismembrator-S and set to shake at 2500rpm for 45 seconds.  The weight of the fine bone powder was recorded and it was stored for RNA extraction. 1ml of Trizol Reagent (Ambion) was added per 125mg of pulverized tissue (typical tissue amounts 350-500mg); samples were incubated in Trizol reagent for 10 minutes at room temperature.  Samples were centrifuged at 500g for 5 minutes at 4˚C, the supernatant was removed carefully to ensure no debris was transferred. 0.3ml of chloroform was added to each 1ml of Trizol reagent, the sample was thoroughly mixed and allowed to incubate at room temperature for 5 minutes.  The samples were centrifuged at 12,000g for 20 minutes at 4˚C.  The colourless upper phase was collected and transferred to a separate tube, to which an equal volume of 70% ethanol was added and incubated at room temperature for 10 minutes.    The samples were then applied to spin cartridges from TRIzol Plus RNA purification kit (Ambion) following the manufactures instructions using the optional On-Column PureLink DNase (Invitrogen) treatment step.  Samples were quantified using spectrophotometry (NanoDrop Thermo) and quality measure using a Bioanalyzer (Agilent).  Only RNA with a RNA integrity number (RIN) above 8 was stored in -80 ˚C and used for RNA-Seq. Samples used for analysis were one long bone and skull pair pooled from two rats, two long bone and skull pairs each pooled from 3 mice, and 3 long bone and skull pairs each from individual macaques.  " cDNA library preparation and sequencing were performed by Eurofins Genomic (Ebersberg, Germany). From the total RNA sample, poly(A)+ RNA was enriched and randomized primer was used for first strand cDNA synthesis.
GEO Accession
GSM4594471