Characterizing role of striated-muscle specific ribosomal protein L3-like (Rpl3L) in mouse hearts

Overall goal: To elucidate the function served by Rpl3L in heart. Purpose of analysis: To determine effects of Rpl3L-deficiency on translational mRNA profile in heart. Experimental structure: Polysomes and monosomes were fractionated from cardiac ventricles and associated RNA extracted for library generation and differential gene expression analyses between wild-type and Rpl3L-null samples, using standard RNAseq pipeline. Overall design: Whole ventricles from wild-type control and Rlp3L-null experimental hearts were pulverized and subsequently manually homogenized using Dounce homogenizers on ice in polysome lysis buffer (10 mM Tris-HCl at pH 7.5, 100 mM KCl, 5mM MgCl2, 0.5% w/ve sodium deoxycholate, 1% v/v IGEPAL CA-630, 10 mM DTT, 150 µg/ml cycloheximide, 1U/µl SUPERase In RNase inhibitor, and 1X HALT protease inhibitor in UltraPure molecular grade H2O). After serial centrifugation, supernatant from final 5-min spin at 16,000 x g at 4°C was layered on top of a 15 to 60% sucrose gradient column and centrifuged at 38,000 RPM for 125 min in a Optima XPN-90 ultracentrifuge (Beckman Coulter + SW 60 Ti swing bucket rotor). Total RNA from monosomal+polysomal fractions was extracted and subjected to downstream sequencing and bioinformatics analyses.