Gel matrix density regulates the expression profile/phenotype of cultured adipocytes
After a 14 day culture on 3%, 5%, or 10% matrix gel, the sample was rinsed with PBS and digested using 250 U/mL Collagenase IV for 45 minutes at 37C. The sample was then centrifuged and the solution was aspirated from the pellet. RNA from the sample was isolated using the QIAGEN QIAshredder and RNeasy mini kit (QIAGEN, Hilden, Germany). Quality assessment was performed using the BioAnalyzer (Agilent Inc., Santa Clara, CA, USA) and RNA concentration measured using a ThermoScientific NanoDrop 2000 Spectrophotometer. Samples were stored at -80C prior to RNA-sequencing. RNA-sequencing was performed by preparing libraries with the SMART-Seq v4 PlUS kit (Takara Bio, Ann Arbor MI, USA) from 2ng of RNA using 11 cycles of cDNA amplification and 15 cycles of library amplification per manufacturer's protocols. Final libraries were checked for quality and quantity by Qubit hsDNA (ThermoFisher Scientific, Inc., Waltham, MA, USA) and LabChip (Perkin Elmer, Waltham, MA, USA). Samples were pooled and sequenced on the Illumina NovaSeq S4 paired-end 150 bp, according to manufacturer's recommended protocols. More information about this study can be found here: Ky A, McCoy AJ, Flesher CG, Friend NE, Li J, Akinleye K, Patsalis C, Lumeng CN, Putnam AJ, O'Rourke RW. Matrix density regulates adipocyte phenotype. Adipocyte. 2023 Dec;12(1) 2268261. doi 10.1080/21623945.2023.2268261. Epub 2023 Oct 10. PMID 37815174; PMCID PMC10566443.