Zymo clean concentrator 5 kit

Tendon and muscle total RNA was extracted using commercially available kits and the TRIzol isolation system for fibrous tissue. Briefly, tissue was manually homogenized via disruption with mortar and pestle. 1 ml of TRIzol Reagent (Life Technologies, Carlsbad, CA, USA) was added for cell lysis and dissociation of nucleoprotein complexes. Homogenized samples were centrifuged for 10 minutes at 4°C at 12,000xg. Supernatant was transferred to a new tube, and 100μl 1-bromo-3-chloropropane was added for phase separation. The aqueous phase where the RNA resides was transferred to a new tube. Molecular grade ethanol was added to the sample. Tendon RNA solutions were cleaned using a Zymo Clean & Concentrator-5 Kit (Zymo, Irvine, CA, USA) with addition of DNAse I (Qiagen, Valencia, CA, USA). Concentrated tendon RNA was eluted using 12 μl DNAse/RNAse-free water. Muscle RNA solutions were loaded into a Zymo DirectZol kit (Zymo, Irvine, CA, USA) and the kit protocol with addition of DNAse I (Zymo, Irvine, CA, USA) was followed. Concentrated muscle RNA was eluted using 25μl of DNAse/RNAse-free water. RNA was stored at -80°C.

100 µg RNA was biotinylated using MTSEA biotin-XX linker (Biotinum, Cat.# 90066) and purified by μMACS streptavidin MicroBeads (Miltenyi Biotec, 130-092-948) as described by Gregersen et al.^{60 (link)}. with the following modifications. The µColumn (Miltenyi Biotec, 130-092-948) was washed three times with 1 ml of 65 °C pre-warmed pull-out wash buffer (100 mM Tris-HCl pH 7.5, 10 mM EDTA, 1 M NaCl, 0.1% TWEEN20 (v/v)), followed by three washes with 1 ml pull-out wash buffer at room temperature. The eluted RNA was purified using the ZYMO Clean & Concentrator-5 kit (ZYMO Research, Cat.# R1013).