GSM9523054: WT control, Established rep 5; Mus musculus; RNA-Seq
Tail tendon fascicles were placed in 2mL Eppendorf Safe-Lock tubes, snap frozen in liquid nitrogen and stored at -80C until further assayed. On the day of RNA isolation, tubes were placed immediately in dry ice. Samples were transferred to supercooled Spex microvial cylinder (6757C3, SPEX™ SamplePrep) containing 150µl of GENEzol™ reagent (GZR200, Geneaid). Samples were cryo-grinded in a bath of liquid nitrogen using FreezerMill (6870, SPEX™ SamplePrep) until fascicles were completely disrupted. Pulverized samples were collected by rinsing the tubes with 900µl GENEzol™, transferred to a clean 1.5 ml tube and kept in dry ice. To purify RNA, tissue homogenate was thawed and mixed well with 200µl of Chloroform (102445, Merck) at a mixing ratio of 1:5. Samples were then spun down for 15min (at 15,000g, 4°C). The upper RNA-containing aqueous phase was transferred to a clean 1.5 ml tube and mixed with one part of 70% ethanol. RNA cleanup was subsequently performed using PureLink™ RNA Micro Scale Kit (12183016, Invitrogen™), including an on-column DNA digestion step with DNase I (DNASE70, Sigma-Aldrich). rRNA depletion was performed using NEBNext rRNA Depletion Kit (Human/Mouse/Rat)._x000D_ RNA sequencing library preparation used NEBNext Ultra RNA Library Prep Kit for Illumina by following the manufacturer's recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented according to manufacturer's instruction. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR.. Sequencing libraries were validated using NGS Kit on the Agilent 5300 or 5600 Fragment Analyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 4.0 Fluorometer (Invitrogen, Carlsbad, CA).