title: GSM4678170: OT-rich bone oim/oim rep2; Mus musculus; RNA-Seq
library_construction_protocol: Femora and tibiae were harvested from WT, oim/oim, and CrtapKO mice at 3 months of age, N=4/genotype. Osteocyte-enriched bone was obtained by removing the epiphyses, thoroughly flushing the bone marrow, and scraping off the periosteal membrane; all these procedures were performed on ice in less than 10 minutes after the initial harvest. The osteocyte-enriched bone was immediately homogenized in 1 mL of TriPure Isolation Reagent (Roche REF 11667157001) and processed for RNA extraction according to the manufacturer's instructions until the chloroform separation step. An equal volume of freshly-made, RNase-free 75% ethanol was added to the clear supernatant obtained from the chloroform separation, mixed, and then transferred to an RNeasy Micro column (Qiagen cat# 74004). Digestion with DNase I and elution of RNA was performed according to the manufacturer's instructions. RNA samples were tested for quality using an Agilent 2100 Bioanalyzer. Only RNA samples with an RNA integrity number (RIN) of ≥7.0 were accepted for sequencing. Sequencing libraries were prepared from the RNA samples by use of the TruSeq mRNA Sample Prep Kit (Illumina). Briefly, total RNA (1μg) was subjected to polyA selection, then chemically fragmented and converted to single-stranded cDNA using random hexamer-primed reverse transcription. The second strand was then generated to create double-stranded cDNA, followed by fragment end repair and addition of a single A base on each end of the cDNA. Adapters were ligated to each fragment end to enable attachment to the sequencing flow cell. The adapters also contained unique index sequences that allowed the libraries from different samples to be pooled and individually identified during downstream analysis. Library cDNA was PCR amplified and enriched for fragments containing adapters at each end to create the final cDNA sequencing library. Libraries were validated on the Fragment Analyzer for fragment size and quantified by use of a Qubit fluorometer (Life Technologies). Equal amounts of each library were pooled for sequencing on the NextSeq 500 platform using a high output flow cell to generate approximately 25 million 75-base reads per sample.
attributes.tag: GEO Accession
attributes.value: GSM4678170