GSM9685622: Dysf mouse rep 2 (6 month old, female); Mus musculus; RNA-Seq
Quadriceps muscles were removed from Dysf and DysfA6 female mice (n=2 each, 4 mice total) and longitudinally cut along the orientation of the myofibers into smaller pieces and digested in in 6 mg/mL of collagenase type II (LS004177; Worthington) solution in 1:5 weight to volume ratio and enzymatically digested at 37°C for 2.5 hours with shaking.  Digested tissues were filtered through 70µm nylon filter to enrich for myofibers.  Remaining tissues on the filter were washed with DPBS with calcium and magnesium (MT21030CV; Thermo Fisher Scientific).  The suspension was then filtered through 100µm nylon filter.  Filtered isolated myofibers were pelleted and flash frozen in liquid nitrogen until RNA isolation.  RNA was isolated using TRIzol Reagent (15596018; Thermo Fisher Scientific) followed by Aurum Total RNA Mini Kit (7326820; Bio-Rad).  Briefly, the frozen myofiber pellet was treated with 1mL of TRIzol Reagent (15596018; Thermo Fisher Scientific) and combined with 200µL of chloroform.  The aqueous phase was combined with equal volume of 70% ethanol and used as starting material for RNA isolation using the Aurum Total RNA Mini Kit following manufacturer's instructions. RNA quality and quantity were assessed by Bioanalyzer RNA pico chip and Qubit RNA HS assay kit, respectively. Then, 25ng of RNA was used as input for library construction using Illumina stranded mRNA prep kit (Illumina 20040534) according to manufacturers' protocol.  The multiplexed libraries were sequenced on Novaseq X Plus using single end 50nt mode.