Computational protocol: Targeting β1-Integrin Signaling Enhances Regeneration in Aged and Dystrophic Muscle in Mice

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Protocol publication

[…] Images of hematoxylin and eosin stained muscle sections were captured from a Nikon 800 microscope with 10× or 20× Plan Apo objectives and Canon EOS T3 camera using EOS Utility image acquisition software. Fluorescent images of muscle sections and single myofibers were wither captured using Leica SP5 confocal equipped with 40×/1.25 Plan Apo oil objectives using Leica image acquisition software, or a Zeiss Axioskope equipped with a 40×/0.5 Plan Apo oil objective and Axiocam camera using Zeiss image acquisition software. Identical exposure times were used and images were processed and scored with blinding using ImageJ64. If necessary, brightness and contrast were adjusted for an entire experimental image set. For quantification of polarized cell markers (Par3, m-cadherin, pp38), all authors individually scored each set of images with blinding using ImageJ64, only those that were agreed upon by all were included. For the rest, Imaris (Bitplane) was used for three-dimensional rendering of fluorescence data for quantification of polarization. A subset was also randomly selected for quantitative analysis via Imaris to confirm authors’ scoring. Cell number, fiber diameter, fiber number, and fiber cross sectional area were determined using ImageJ64 or Fiji using images of a micrometer (VWR) taken under the same magnifications as the sample images as references for imaging field sizes. [...] For RNA-seq analysis, FACS isolated SCs were cultured in minimal growth media on Matrigel-coated plates. At 24, 48 and 72 h, RNA was isolated using the Arcturus PicoPure RNA isolation kit (Applied Biosystems). The Ovation RNA-Seq System (NuGEN) was used to prepare amplified cDNA. Libraries for single-end sequencing were prepared using Illumina’s TruSeqDNA sample prep kit LT. Sequencing was carried out on an Illumina HiSeq2000 to generate single-end 100bp reads, which were aligned to the mouse genome (mm9) using TopHat (v2.0.7). Cufflinks (v2.1.1) was used for differential expression analysis. Only genes that displayed “yes” significance as determined by Cuffdiff 2 were analyzed further: if P value of observed change in FPKM was greater than the FDR (False discovery rate) after Benjamini-Hochberg correction for multiple-testing. […]

Pipeline specifications

Software tools TopHat, Cufflinks
Application RNA-seq analysis
Organisms Mus musculus
Diseases Muscular Diseases, Muscular Dystrophy, Duchenne