Cfx96 touch real time system thermocycler

Reverse Transcription was performed using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific). Gene expression was quantified with TaqMan Expression assays on a CFX96 Touch Real-Time System thermocycler (Bio-Rad). Gene expression assays were normalized to the expression of RPS9. Individual gene assay IDs: CSGALNACT1 Hs00218054_m1, VCAN Hs0017642_m1, CHST3 Hs01000045_m1, CHST11 Hs00218229_m1, RPS9 Hs02339424_m1. Expression values reported are averaged across at least three (n = 3) biological replicates unless otherwise stated in the main text.

Total RNA was isolated from tissue or cells using Total RNA Purification Kit (Norgen Biotek) as per manufacturer’s instructions. Reverse transcription was performed using the High-Capacity RNA-to-cDNA Kit (Thermo Fisher Scientific) for gene analysis or using the TaqMan MircoRNA Reverse Transcription Kit (Thermo Fisher Scientific) for microRNA analysis. Gene and microRNA expression were quantified with TaqMan Expression assays on a CFX96 Touch Real-Time System thermocycler (Bio-Rad). Gene expression assays were normalized to the expression of RPS9, and microRNA expression assays were normalized to the expression of RNU6. Individual gene assay IDs include the following: CDH1, hs01023895; DNAJB1-PRKACA, custom; FANCC, hs0098454; KLF11, hs00231614; miR-10b, 002218; miR-21, 000397; miR-182, 002334; PTEN, hs02621230; RNU6, 001973; RPS9, hs02339424; SEC14L2, hs00391446; SIRT5, hs00978331; SUN2, hs00391446; and TRIM35, hs00324633 (Thermo Fisher Scientific). Expression values reported are averaged across at least 3 biological replicates unless otherwise stated in the main text.