alias: E-MTAB-5555:NaiveBone_2_p
title: Differences in the osteocyte-specific transcriptome in the presence of multiple-myeloma tumour burden
design: Differences in the osteocyte-specific transcriptome in the presence of multiple-myeloma tumour burden
library_construction_protocol: Mice were sacrificed by CO2 asphyxiation and cervical dislocation. Both humeri were isolated by disarticulation from 6 mice. Soft tissue including muscle, ligaments and tendons were removed, before the surface was gently scraped with a scalpel to remove the periosteum. Diaphysial bone from the humeri was isolated by cutting immediately proximal to the deltoid tuberosity and then 1mm proximal to the epicondyles before completely removing the deltoid tuberosity along the bone shaft. Bone marrow was removed from the humeri by first flushing with PBS (fraction taken for sequencing) until visibly clean and then centrifuging at 14,000rcf for 15 seconds. Bilateral samples from each mouse were processed and snap frozen together in liquid N2 within ~16 minutes of sacrifice. Bone marrow flushed from each bone (before centrifugation) was resuspended in 1 ml PBS before centrifugation at 800rcf for 5 mins. The PBS supernatant was removed and the pellet resuspended in 1ml of TRIreagent (Sigma T9424) and stored at -20 degrees celsius. All animal experiments were performed in accordance with an approved protocol of the St Vincents Hospital Animal Ethics committee (ARA 14/43).  6-8 week old C57BL/KaLwRijHsd (BKAL) male mice (Harlan, Netherlands) were injected intravenously with 0.5 x 106 syngeneic 5TGM1-eGFP murine myeloma cells in 200 Î¼l PBS49,65. Mice were with calcein (s.c. 20mg/kg) 6 and 2 days prior to harvest. Mice were sacrificed at 21 days (5TGM1) and the bilateral humeri collected for RNAseq analysis. Bone samples: 800 ml TRIreagent (Sigma T9424) was added directly to the frozen bone samples and homogenised using a Polytron hand held homogeniser (Kinematica PT 1200 E 11010026). RNA was isolated according to the manufacturers protocol and cleaned with an additional ethanol-precipitation step. Bone marrow samples: RNA was isolated according to the manufacturers protocol. All samples: RNA yield was determined using a Nanodrop (Thermo) and RNA integrity determined using the Bioanalyser RNA 6000 Nano Kit (Agilent Technologies 5067-1511). Total-RNA (200ng) was depleted of ribosomal RNA using RNaseH (Epicentre H39500) and ribosomal RNA targeting oligonucleotides based on a protocol by Adiconis et al., 2013 (Adiconis et al., 2013). Briefly, total RNA, spiked with ERCC internal controls (Life Technologies 4456739), was incubated with ribosomal-RNA targeting oligos and RNaseH to degrade the rRNAs before the oligos were removed with a DNase treatment (Life Technologies AM2238). RNA was repurified using magnetic beads according to the manufacturers protocol (Beckman Coulter Genomics A63987). Total-RNA, 125bp paired-end, stranded transcriptome libraries were prepared using the TruSeq Stranded Total RNA LT Sample Prep Kit starting from the fragmentation step in the manufacturers protocol (Illumina RS-122-2201).
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-5555:NaiveBone_2_p
attributes.tag: Experimental Factor: normal
attributes.value: disease
attributes.tag: Experimental Factor: bone
attributes.value: organism part