title: Illumina HiSeq 4000 sequencing; RNA sequencing of native and ex vivo cultured murine tail tendon fascicles
design: RNA sequencing of native and ex vivo cultured murine tail tendon fascicles
library_construction_protocol: 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.