GSM5838188: RNA-Seq, AAV9-treated; Mus musculus; RNA-Seq
Frozen muscle biopsies (~ 4 milligrams) were cut on a cryostat (20 microns) and homogenized with a 26-gauge needle in 400 microliters of 20 mM Tris*Cl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/ml cycloheximide and 1% Triton X-100.  Ribosome protected fragments were isolated from one half of the homogenate as previously described (N. Wein et al.,(2014). Translation from a DMD exon 5 IRES results in a functional dystrophin isoform that attenuates dystrophinopathy in humans and mice. Nature medicine 20: 992-1000). RNA was extracted using TRIzol® and precipitated with isopropanol according to the manufacturer's instruction (Life Technologies). Illumina TruSeq Stranded Total RNA library kits were used to prepare RNA-Seq indexed libraries and 50 bp reads were generated on an Illumina HiSeq instrument using v4 chemistry.  RPF-Seq libraries were prepared using the Illumina TruSeq Small RNA Sample Kit according to the manufacturer's specifications and 50 bp reads were generated on an Illumina HiSeq instrument.