GSM5939179: BAT, totalRNA1; Mus musculus; RNA-Seq
For sample collection of BAT/WAT/Beige cultures for Ribo-Seq and total mRNA-Seq used for smORF discovery, each culture was washed directly out of the incubators 3x with ice-cold PBS supplemented with 100μg/mL cyclohexamide (Fisher Scientific #AAJ66004X). After the last wash, liquid nitrogen was gently ladled onto the surfaces of the cells and plates were stored at -80 °C prior to Ribo-Seq analysis. Each biological replicate for Ribo-Seq in our experiments represents 11 wells of a 6-well dish leaving one well to be lysed with Trizol reagent (Thermo #15596026) for the bulk mRNA-Seq used int the de novo transcriptome assembly of the cells under analysis. Cells were lysed with 400 μl of ice-cold lysis buffer (20 mM Tris–HCl, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1% Triton X-100, with 1 mM DTT, 25 U ml−1 Turbo DNase (Thermo Fisher, catalog no. AM2238) and 100 μg ml−1 CHX added fresh) was dripped onto the plate. Cells were incubated on ice in lysis buffer for 10 min with periodic vortexing and pipetting to disperse the cells. The lysate was then clarified by centrifugation at 15,000g for 10 min. Cell lysates were flash-frozen in liquid nitrogen and stored at −80 °C for up to 7 d before ribosome footprinting. For each cell type, ribosome footprinting was carried out by digesting 40-60 μg of RNA in 200-300 μL lysate with 0.375 U/μg−1 RNase I (Lucigen, N6901K) for 50 min at room temperature. Digestion reactions were quenched with 200 U Superase-In RNase I inhibitor (Thermo Fisher, AM2694) on ice. Following digestion, monosomes were purified from small RNA fragments using MicroSpin S-400 HR columns (GE Life Sciences), and ribosome protected RNA fragments (RPFs) were extracted by acid phenol chloroform and isopropanol precipitation.  TruSeq mRNA and Total RNA kits (Illumina) were used to prepare polyA and total RNA libraries. total RNA and polyA mRNA paired end deep sequencing