1 bromo 3 chloropropane extraction

The preparation of frozen AF and NP tissue samples was performed under permanent cooling on dry ice. When macroscopically visible, other tissues like the cartilage endplate, bony endplate or ligamentum flavum were carefully removed. All AF and NP samples first underwent haptic and macroscopic evaluation. Second, a representative cross-section of each sample was taken for histological analysis.
The remaining tissue sample was used for RNA isolation. The frozen tissue was minced and immediately lysed and homogenised in TriReagent (Sigma-Aldrich, Taufkirchen, Germany) using a TissueRuptor (Qiagen, Hilden, Germany). Total RNA was isolated by 1-bromo-3-chloropropane extraction (Sigma-Aldrich) and subsequent purification using the RNeasy Mini Kit including on-column DNase I digestion (Qiagen) according to the manufacturer’s instructions. The RNA quality was determined by measuring RNA integrity number (RIN) value with RNA 6000 Pico Assay using the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). Only samples with RIN > 6.0 were used for analysis.

Native human AF tissues of three mild and three severe degenerated IVDs were snap frozen in liquid nitrogen and stored at −80 °C until ribonucleic acid (RNA) isolation. Cell isolation from native AF tissues of three mild and three severe degenerated IVDs was performed by overnight digestion using a collagenase mix as described earlier [19 (link)]. Cells were seeded with a density of 10,000 cells/cm² and cultivated up to passage 2. Cell lysis of the tissue was performed using Tri-Reagent (Sigma-Aldrich, Taufkirchen, Germany). For native tissue, the tissue was minced in Tri-Reagent using the Qiagen TissueRuptor (Qiagen, Hilden, Germany). After 1-bromo-3-chloropropane extraction (Sigma-Aldrich), further RNA isolation was performed using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany) according the manufacturer’s protocol.