Truseq ribo profile mammalian library prep kit

Ribosome profiling was performed using the TruSeq Ribo Profile (Mammalian) Library Prep Kit (Illumina #RPHMR12126) according to the manufacturer’s protocol. The Illumina Ribo-Zero Gold rRNA Removal Kit (H/M/R) (Illumina #MRZG12324) was employed to deplete ribosomal RNA samples. The library quality was verified using a Bioanalyzer. Libraries were sequenced using the NextSeq™ 500 High Output Kit (FC-404-1005) on a NextSeq 500 platform (Illumina) in a 75-bp single read run.

Sample preparation for ribosome profiling was conducted according to the manufacturer’s specifications for the TruSeq Ribo Profile (Mammalian) Library Prep Kit (Illumina). Briefly, HEK293 cells were treated with CHX (Sigma-Aldrich, final concentration 0.1 mg/ml) for 1 min. In-dish cell lysis was performed using mammalian lysis buffer (including CHX at a concentration of 0.1 mg/ml). Then 600 μl of lysate were taken and 15 μl of RNase I (100 U/μl, Thermo Fisher Scientific) were added and the mixtures were incubated for 45 min at room temperature, followed by adding 15 μl SUPERaseIn RNase Inhibitor (Ambion, Thermo Fisher Scientific) to stop the reaction. Ribosome recovery was performed by illustra MicroSpin S-400 HR Columns (GE Healthcare) and the RPFs were purified by RNA Clean & Concentrator (Zymo Research). Ribosomal RNAs were depleted using Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat, Illumina). RPFs without ribosomal RNA were run on a 15% urea denaturing-PAGE gel, and the gel slices corresponding to 28–30 nts were excised. The RPF RNAs were eluted and precipitated followed by library construction according to the manufacturer’s protocol.

Approximately 8–9 × 10⁶ ESCs (per sample) were treated with cycloheximide (0.1 mg/ml; Sigma, 01810–1 G) for 1 min prior to trypsinization and cell lysis. Control and NF-YA KD cells were used as input material for the TruSeq Ribo Profile Mammalian Library Prep Kit (Illumina) following the manufacture’s protocol.