Illumina HiSeq 2500 paired end sequencing; The osteocyte transcriptome during post-natal skeletal maturation in male and female mice
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 200ÂµL 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).