GSM8989040: 3T3-L1 differentiated Day 7, biol rep 1, Riboseq; Mus musculus; RNA-Seq
Cells were washed twice after treatment with ice cold PBS supplemented with 100ug/mL cycloheximide (CHX; Fisher Scientific, AAJ66004X) and snap frozen in liquid nitrogen and stored at -80°C. After thawing briefly on ice, the cells were lysed with 300uL of ice-cold lysis buffer (20mM Tris-HCl, pH 7.4, 150mM NaCl, 5mM MgCl2, 1% Triton X-100, 1mM DTT, 25U Turbo DNase (Thermofisher Cat# AM2238), and 100ug/mL CHX – added fresh) for 10 minutes with periodic vortexing and pipetting through an 18G needle to ensure the cells are dispersed. Cell lysates were clarified by centrifugation for 15,000g for 10 minutes at 4°C, flash frozen in liquid nitrogen, and stored in -80°C for up to 1 week. For each 3T3-L1 cell condition, ribosome footprinting was carried out by digesting 50 ug of RNA in 300 uL lysate with 0.375 U/ug RNase I (Lucigen, N6901K) for 45 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 Micro-Spin S-400 HR columns (GE Life Sciences), and ribosome protected RNA fragments (RPFs) were extracted by acid phenol chloroform and isopropanol precipitation. Sequencing libraries were prepared as in McGlincy and Ingolia with some modifications. First, the RiboZero Mammalian Kit (Illumina) was used to deplete rRNA after RPF extraction and just prior to RPF size selection by gel extraction. Second, the Zymo clean & concentrator step after adaptor ligation is omitted and the reaction was carried over straight into reverse transcription. For the reverse transcription step to form cDNA, Episcript RT (Lucigen, ERT12910K) was used. Following reverse transcription, excess primer was degraded using Exonuclease I (Lucigen, X40520K) and the RNA templates were degraded using Hybridase (Lucigen, H39500). For the cDNA circularization step, CircLigase I (Lucigen, CL4111K) was used. PCR amplification was then carried out using Phusion Hot Start II High-Fidelity Master Mix (Thermo Fisher, F565L) for 9–12 cycles.