title: The clock transcription factor BMAL1 is a key regulator of extracellular matrix homeostasis and cell fate in the intervertebral disc
design: The clock transcription factor BMAL1 is a key regulator of extracellular matrix homeostasis and cell fate in the intervertebral disc
library_construction_protocol: For circadian time series whole lumbar IVDs from 2-3-month-old male and female WT and Col2a1-Bmal1-/- mice were collected 4 hours apart over a period of 48 hours, starting at 9 am. Two biological replicates were obtained per genotype and timepoint. Whole lumbar IVDs were resected, and paraspinal tissues carefully removed. Tissues were immediately snap frozen and stored at -80oC for later processing. For AF tissue RNAseq male and female three-month-old WT and Col2a1-Bmal1-/- mice were collected at 8 am. Three biological replicates were obtained per genotype and timepoint. Tail discs were resected, and the NP and AF were manually separated under a dissection microscope to ensure careful separation of tissue. AF tissues were kept on ice and kept hydrated in HBSS throughout the dissection procedure, then snap frozen and stored at -80oC for later processing. All animal studies were performed in accordance with the 1986 UK Home Office Animal Procedures Act. Approval was provided by the local ethics committee. Mice were maintained at 20-22°C, with standard rodent chow available ad libitum and under 12:12 hr light dark schedule (light on at 7 am; light off at 7 pm). The PER2::Luc mice carry the firefly luciferase gene fused in-frame with the 3' end of the Per2 gene, creating a fusion protein reporter (Yoo et al. PNAS 2004). Bmal1flox/flox - PER2::Luc mice were subsequently crossed with Col2a1-Cre mice expressing Cre recombinase under the control of the Col2a1 promoter3 to generate IVD /cartilage specific Bmal1 KO. All mice were bred in-house at the University of Manchester.  Generation and genotyping of the Col2a1-Bmal1-/- mice was described before (Dudek et al. JCI 2016) For the isolation of RNA tissues were homogenised in TRIzol using a Mikro-Dismembrator S (Satorius Stedim Biotech) at 2000 rpm for 30 seconds, twice. Liquid nitrogen was used to keep tissues and homogenates cold at all times during the homogenisation process. Homogenates were subsequently processed using 1-Bromo-3-chloropropane to separate RNA. The aqueous phase was processed twice to ensure clean separation of RNA from other organic matter. Glycogen was used to capture mRNA. Pellets were washed three times in 70% EtOH, dried and resuspended in RNAse-free H2O. The quantity of resuspended mRNA was measured using a QubitTM 4 Fluorometer (Thermo Fisher Scientific) and RNA integrity measured using a 2200 TapeStation (Agilent Technologies) to ensure good quality for downstream analysis. Libraries were generated by the Genomic Technologies Core Facility using the TruSeq® Stranded mRNA assay (Illumina, Inc.) according to the manufacturer's protocol