Illumina HiSeq 4000 sequencing; RNA sequencing of native and ex vivo cultured murine tail tendon fascicles
RNA sequencing of native and ex vivo cultured murine tail tendon fascicles
Tail tendon fascicles were extracted from mice straight after euthanasia and either snap frozen in liquid nitrogen or cultured for 6 days in either serum-free or serum containing medium (10%) at 37Â°C and atmospheric O2 saturation. 16 to 20 fascicles were pooled and snap frozen before RNA extraction.  Samples were pulverized in QIAzol lysis reagent (#79306, Qiagen) by cryogenic grinding (FreezerMill 6870, SPEXTMSamplePrep). Tissue lysates were mixed with 1-bromo-3-chloropropane (B9673, Sigma-Aldrich) at a ratio of 1:4 and the RNA containing aqueous/organic phase was separated using 5Prime PhaseLock Gel Heavy (#2302830, LabForce). RNA was extracted by using the RNeasy micro Kit (74004, Quiagen). The quantity and quality of the isolated RNA was determined with a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). A total RNA amount of 100ng was used with the TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc, California, USA) for library preparation.