alias: E-MTAB-5533:Marrow_4_p
title: Illumina HiSeq 2500 paired end sequencing; Identifying genes enriched for expression in osteocytes: transcriptome sequencing mouse bone with and without bone marrow.
design: Identifying genes enriched for expression in osteocytes: transcriptome sequencing mouse bone with and without bone marrow.
library_construction_protocol: Mice were sacrificed by CO2 asphyxiation and cervical dislocation. Both humeri were isolated by disarticulation from 5 mice (a total of 10 bone samples). Soft tissue including muscle, ligaments and tendons were removed, before the surface was gently scraped with a scalpel to remove the periosteum. Diaphyseal 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 left humeri by first flushing with PBS until visibly clean and then centrifuging at 14,000rcf for 15 seconds(\"Clean_bone\" samples), whilst the marrow was retained in right humeri (\"Bone_and_marrow\" samples). Bones were cut into pieces and snap frozen in liquid N2 prior to homogenisation and RNA extraction. All samples from each mouse were processed and snap frozen in liquid N2 within ~16 minutes of sacrifice. 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. 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 (250ng) 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: Garvan Institute of Medical Research
identifiers.id: E-MTAB-5533:Marrow_4_p
attributes.tag: Experimental Factor: bone and bone marrow
attributes.value: sampling site