Computational protocol: RNA helicase, DDX27 regulates skeletal muscle growth and regeneration by modulation of translational processes

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Protocol publication

[…] Functional experiments were performed as previously described []. Briefly, fish were studied in a bicarbonate buffer of the following composition: (in mM) 117.2 NaCl, 4.7 KCl, 1.2 MgCl2, 1.2 KH2PO4, 2.5 CaCl2, 25.2 NaHCO3, 11.1 glucose (Dou et al., 2008). 4–5 dpf larvae were anesthetized in fish buffer containing 0.02% tricaine and decapitated. The head tissue was used for genotyping. The larval body was transferred to a small chamber containing fish buffer equilibrated with 95% O2, 5% CO2 and maintained at 25°C. The larval body was attached to an isometric force transducer (Aurora Scientific, Aurora, Ontario, CAN, model 403A) and position motor (Aurora Scientific model 308B) using a 10–0 monofilament tie placed at the gastrointestinal opening and another tie attached several myotomes proximal from the tip of the tail. Twitches (200 μs pulse duration) and tetani (300 Hz) were elicited using supramaximal current delivered to platinum electrodes flanking the preparation. All data were collected at the optimal preparation length (Lo) for tetanic force. At the conclusion of the experiment, images of the preparations width and depth at Lo were obtained by carefully rotating the preparation about the gastrointestinal opening attachment point. Each image was analyzed by ImageJ using an internal length calibration. Preparation cross-sectional area (CSA) was calculated from width and depth measurements assuming the preparations cross-section was elliptical. Forces were calculated as active force, i.e. peak force minus the unstimulated baseline force, and are presented in absolute terms (valid because all larvae were attached at a consistent anatomical landmark, the gastrointestinal opening) as well as normalized to preparation CSA. The maximal rate of twitch tension development was determined as the maximal derivative of the force by time response between the onset of contraction and peak force. Likewise, the maximal rate of twitch tension relaxation was calculated as the first derivative of the force by time relaxation response, ranging from peak force until force had declined to approximately baseline. Statistical differences in the ddx27 (n = 12) and control (n = 10) group means were evaluated by a two-sample t-test. [...] Polysome ribosome fractions were prepared from control and Ddx27 C2C12 myoblasts. Equal number of control and Ddx27 knockout C2C12 myoblasts were plated in 10 cm dishes in the proliferation media and collected after 24 hours for polysomal analysis. Polysomes were isolated as described above for zebrafish muscle and fractions were pooled together, treated with proteinase K and RNA was isolated using acid phenol-chloroform extraction and ethanol precipitation. As Ddx27 myoblasts exhibit a reduced proliferation, equal amounts of proteins were used to fractionate polysomes from control and KO myoblasts. Deep sequencing libraries were generated and sequenced as described []. Ribosomal profiling was repeated in triplicate and principal component analysis was performed to identify variation between samples. The Spearman R2 was > 0.9 for all replicates (except one sample) that was subsequently removed from follow up analysis. Splice-Aware alignment program STAR was used to map sequencing reads to Mus musculus (mm10 build). R package “edgeR” was employed to identify differential gene expression calls from these sequence reads. Gene expression was considered to be up-regulated if log2FC> +1 or downregulated if the log2FC< -1 (FC = fold change of average CPM) with respect to the condition being compared at a false discovery rate <0.05. Functional ontological classification of different gene lists was performed by DAVID. […]

Pipeline specifications

Software tools ImageJ, edgeR
Application Microscopic phenotype analysis