title: Female4_3
organism: Mus musculus
description: Protocols: Mice were sacrificed by CO2 asphyxiation and cervical dislocation. The tibiae, femora and humeri were isolated by disarticulation from 8 mice (a total of 32 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 tibia was isolated by cutting the tibia at the fibulae junction and 1mm distal to the proximal growth plate, and from the femur by cutting the bone immediately proximal to the third trochanter and 1mm proximal to the distal growth plate. The humeri were cut 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 from each bone was removed by first flushing with PBS until visibly clean and then centrifuging at 14,000rcf for 15 seconds. Bones from the left side were cut into pieces and snap frozen in liquid N2 prior to homogenisation and RNA extraction. Calvaria samples were collected by isolating the parietal region of the skull, excluding the sutures and gently scraping soft tissue from the bone surface. Calvaria were separated into left and right bone fragments. Bone samples were cut into 1mm strips and vortexed in a 1.5mL centrifuge tube with 200µL of PBS and then centrifuged at 14,000rcf for 15 seconds to remove bone marrow. Bones from the left side were snap frozen in liquid N2 prior to homogenisation and RNA extraction. Both samples were snap frozen for RNA isolation. 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).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2018-12-31T17:02:31Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-11-30T15:55:55Z
attributes.tag: External Id
attributes.value: SAMEA5145310
attributes.tag: INSDC center name
attributes.value: Garvan Institute of Medical Research
attributes.tag: INSDC first public
attributes.value: 2018-12-31T17:02:31Z
attributes.tag: INSDC last update
attributes.value: 2018-11-30T15:55:55Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7447:Female4_3
attributes.tag: age
attributes.value: 4
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: pre-puberty
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: individual
attributes.value: 3
attributes.tag: organism part
attributes.value: bone
attributes.tag: sample name
attributes.value: E-MTAB-7447:Female4_3
attributes.tag: sampling site
attributes.value: humerus
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: C57BL6/NTac