biostudies-arrayexpressDonald DunbarMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-16564Comparions of transcriptomes of two osteocyte cell lines under conditions that increase and decrease androgens. To study gene expression differences and the cell lines' appropriateness for studying androgen regulation of skeletal functionbiostudies-arrayexpressNucleic Acid Extraction - Cells were collected by scraping into ice-cold PBS and collected by centrifugation (5 minutes at 300 x g; 4°C). Total RNA was extracted using TRIzol reagent (Invitrogen Paisley, UK; #15596026) as per manufacturer’s instruction. Samples from mouse tibia were extracted in Qiazol (Qiagen, Manchester, UK; #79306) and homogenised on ice for 3 x 10 seconds using a rotor-stator homogeniser (Ultra-Turrax T8). RNA was purified using the Qiagen RNease Mini Kit (Qiagen, #74104) and RNA concentration and RIN integrity was determined via TapeStation (Agilent 2200, Agilent Technologies). Only samples with RIN values ≥ 6 (tissue) and ≥8 (cells) respectively were used in downstream analysis. Total RNA was reverse transcribed using the High-Capacity RNA-to-cDNA kit (Applied Biosystems, Birchwood, UK; #4387406);Growth Protocol - MLO-A5 and MLO-Y4 were routinely cultured on collagen coated plates in control medium (Shah et al., 2016): alpha minimum essential media (αMEM; #MEM-A) supplemented with 5% (v/v) heat-inactivated foetal bovine serum (HI-FBS, #FBS-12A), 5% (v/v) heat-inactivated calf serum (HI-CS, #CS-1A), and 100 U/mL (v/v) penicillin and 100 µg/mL streptomycin (#PS-B). To simulate androgen deprived (AD) conditions, phenol red free αMEM (PRF-αMEM) was used in place of αMEM and media was supplemented as above plus 2 mM L-glutamine (#GLN-GB). LNCaP cells were cultured in prostate cancer control medium: Roswell Park Memorial Institute (RPMI) 1640 culture medium (Gibco, Paisley, UK; 21870076), supplemented with 10% HI-FCS, 2 mM L-glutamine, and 100 U/mL (v/v) penicillin and 100 µg/mL streptomycin. To simulate AD conditions, cells were cultured in phenol red free RPMI (PRF-RPMI) (Gibco; 32404014) supplemented with heat-inactivated charcoal stripped foetal calf serum (HI-CS-FCS; Sigma, Poole, UK; #F6765). All cells were cultured in a humidified atmosphere of 5% CO2 at 37°C, and passaged by trypsinisation once 80-90% confluent.Library Construction - RNA-Seq analysis was performed by Novogene (Cambridge, UK). RNA-Seq libraries were prepared using the Novogene NGS RNA Library Prep Set (#PT042). RNA (≥ 200 ng) from MLO-A5 or MLO-Y4 cells (n=4) was purified using poly-T oligo-attached magnetic beads then fragmented, reverse transcribed using random hexamer primers and ligated to adapters.Sequencing - RNA-Seq analysis was performed by Novogene (Cambridge, UK). Quantified libraries were pooled and sequenced using the Illumina NovaSeq X Plus Series system, with pair-end 150 bp read length and ≥35 million read pairs per sample.Sample Collection - C57BL/6JCrl mice were maintained in accordance with the Home Office code of practice (for the housing and care of animals bred, supplied or used for scientific purposes). Animal studies were conducted in line with the ARRIVE guidelines and were fed standard laboratory chow and maintained in a 12-hour light/dark cycle, at 21 ± 2°C. Tibia were collected from four-week-old male and female mice immediately after sacrifice. The epiphyses and growth plate were removed before centrifugation at 15,700 xg at 4°C for 30 seconds to separate the bone marrow tissue from the bone. Tibiae were snap frozen in liquid nitrogen and stored at -80°C for RNA extraction (Hsu et al., 2022). Cell lines and culture conditions MLO-A5 and MLO-Y4 cells were a generous gift from Prof. Lynda Bonewald (University of Missouri–Kansas City, USA). LNCaP cells were obtained from the ATCC. All tissue culture supplements were obtained from Capricorn Scientific (Hessen, Germany) unless otherwise stated. MLO-A5 and MLO-Y4 were routinely cultured on collagen coated plates in control medium (Shah et al., 2016): alpha minimum essential media (αMEM; #MEM-A) supplemented with 5% (v/v) heat-inactivated foetal bovine serum (HI-FBS, #FBS-12A), 5% (v/v) heat-inactivated calf serum (HI-CS, #CS-1A), and 100 U/mL (v/v) penicillin and 100 µg/mL streptomycin (#PS-B). To simulate androgen deprived (AD) conditions, phenol red free αMEM (PRF-αMEM) was used in place of αMEM and media was supplemented as above plus 2 mM L-glutamine (#GLN-GB). LNCaP cells were cultured in prostate cancer control medium: Roswell Park Memorial Institute (RPMI) 1640 culture medium (Gibco, Paisley, UK; 21870076), supplemented with 10% HI-FCS, 2 mM L-glutamine, and 100 U/mL (v/v) penicillin and 100 µg/mL streptomycin. To simulate AD conditions, cells were cultured in phenol red free RPMI (PRF-RPMI) (Gibco; 32404014) supplemented with heat-inactivated charcoal stripped foetal calf serum (HI-CS-FCS; Sigma, Poole, UK; #F6765). All cells were cultured in a humidified atmosphere of 5% CO2 at 37°C, and passaged by trypsinisation once 80-90% confluent. Androgen deprivation and androgen supplementation MLO-A5 and MLO-Y4 cells were seeded at 6.7 × 10³ cells/cm² and 5.3 × 10³ cells/cm², respectively, on collagen-coated T-75 flask in control medium. Twenty-four hours later, media was removed and cells were washed with PBS. To simulate androgen deprivation, cells were cultured in AD medium, containing 2.5 μM enzalutamide. To simulate androgen supplementation, cells were cultured in control medium supplemented with 1 nM R1881. MLO-A5 and MLO-Y4 cells were cultured in their respective medium for 7 days, replacing the drug and culture medium every 3 days. LNCaP cells were cultured on poly-D-lysine coated T-75 flask in prostate cancer control medium. To account for the varying growth rates in the presence of different treatments, LNCaP cells were seeded at 3.3 x 104 cells (control plus drug vehicle or androgen supplemented), 1.3 x 104 cells (AD plus drug vehicle), or 2.7 x 104 cells (AD plus enzalutamide). Twenty-four hours later, culture medium was discarded and cells were washed with PBS. To simulate androgen supplementation, LNCaP cells were grown for 48 hours in control medium supplemented with 0.1% DMSO (drug vehicle) or 1 nM R1881. To simulate AD, LNCaP cells were cultured in AD medium containing 0.1% DMSO (drug vehicle) or 10 µM enzalutamide. Cells were grown for a further 7 days, replacing the culture medium and drug on day 3; cells were not passaged during the treatments.Sample Treatment - Androgen deprivation and androgen supplementation MLO-A5 and MLO-Y4 cells were seeded at 6.7 × 10³ cells/cm² and 5.3 × 10³ cells/cm², respectively, on collagen-coated T-75 flask in control medium. Twenty-four hours later, media was removed and cells were washed with PBS. To simulate androgen deprivation, cells were cultured in AD medium, containing 2.5 μM enzalutamide. To simulate androgen supplementation, cells were cultured in control medium supplemented with 1 nM R1881. MLO-A5 and MLO-Y4 cells were cultured in their respective medium for 7 days, replacing the drug and culture medium every 3 days. LNCaP cells were cultured on poly-D-lysine coated T-75 flask in prostate cancer control medium. To account for the varying growth rates in the presence of different treatments, LNCaP cells were seeded at 3.3 x 104 cells (control plus drug vehicle or androgen supplemented), 1.3 x 104 cells (AD plus drug vehicle), or 2.7 x 104 cells (AD plus enzalutamide). Twenty-four hours later, culture medium was discarded and cells were washed with PBS. To simulate androgen supplementation, LNCaP cells were grown for 48 hours in control medium supplemented with 0.1% DMSO (drug vehicle) or 1 nM R1881. To simulate AD, LNCaP cells were cultured in AD medium containing 0.1% DMSO (drug vehicle) or 10 µM enzalutamide. Cells were grown for a further 7 days, replacing the culture medium and drug on day 3; cells were not passaged during the treatments.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - The RNA-Seq data were processed through the nf-core ‘rnaseq’ version 3.14.0 (doi: 10.5281/zenodo.1400710) and nf-core ‘differential abundance’ version 1.5.0 (doi: 10.5281/zenodo.7568000) pipelines. The ‘rnaseq’ pipeline took raw ‘fastq’ sequencing files and performed quality control with FastQC (Andrews, 2010) and collation with MultiQC (Ewels et al., 2016); ribosomal RNA removal with SortMeRNA (Kopylova et al., 2012); and adaptor and quality trimming with Trim Galore (Krueger, 2012) packages. The processed sequences were then aligned to the reference genome (Mus musculus GRCm39, Ensembl release 112) with the STAR package (Dobin et al., 2013) and quantified with the salmon package (Patro et al., 2017), generating a gene expression read counts matrix with a count for each gene in each sample. The ‘differentialabundance’ pipeline took the gene expression matrix and transcript length matrix from the ‘rnaseq’ pipeline output and calculated differential gene expression with the DESeq2 package (Love et al., 2014); and generated tables, plots, and static and interactive reports.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XRNA-seq of coding RNAMus musculusTranscriptomic comparison of osteocyte cell lines - determining their utility as models to study sex hormone regulation of skeletal functionDonald DunbarRachel L Wade, Donald R Dunbar, Gurå T Bergkvist, Colin Farquharson, Jennifer A FraserfalseTranscriptomic comparison of osteocyte cell lines – determining their utility as models to study sex hormone regulation of skeletal functionComparions of transcriptomes of two osteocyte cell lines under conditions that increase and decrease androgens. To study gene expression differences and the cell lines' appropriateness for studying androgen regulation of skeletal function2026-09-01T00:00:00Z2026-09-01T08:13:57.157Z2026-01-23T15:33:43.247ZE-MTAB-16564publ-0-dxq0-removableERP188095EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_000396910.1016/j.gep.2026.119414