GSM9111447: SW1 sh Polr1a biol rep 3; Mus musculus; OTHER
To prepare RFs, 10μL of RNase I and 6μL of DNase I were added to 400μL of lysate， which was then incubated for 45 min at room temperature with gentle mixing on a Nutator mixer. Nuclease digestion was stopped by adding 10μL of SUPERase·In RNase inhibitor. Size exclusion columns (illustra MicroSpin S-400 HR Columns; GE Healthcare; catalog no. 27- 5140-01) were equilibrated with 3 mL of polysome buffer by gravity flow and centrifuged at 600g for 4 min at room temperature. 100μL of digested RFs were added to the column and centrifuged at 600g for 2 min. Next, 10μL 10% (wt/vol) SDS was added to the elution, and RFs with a size greater than 17nt was isolated according to the RNA Clean and Concentrator-25 kit (Zymo Research;R1017). rRNA was removed using short (50–80 bases) antisense DNA probes complementary to rRNA sequences were added to the solution containing RFs, then RNase H and DNase Iwas added to digest rRNA and residual DNA probes. Finally, RFs were further purified using magnet beads. Ribo-seq libraries were prepared using NEBNext® Multiple Small RNA Library Prep Set for Illumina® (catalog no.E7300S, E7300L). Briefly, adapters were added to both ends of RFs, followed by reverse transcription and PCR amplification. The 140-160bp size PCR products were enriched to generate a cDNA library and sequenced using Illumina HiSeqTM X10.