GSM6398470: SARS-CoV-2_variant_omicron_N_RNA-SELEX_cycle3_rep3; Escherichia coli; SELEX
The pellet was suspended in 10 ml buffer A (0.5 mg/ml Lysozyme, 50 mM tris-HCl pH 7.5, 300 mM NaCl, 10 mM Imidazole, 1 mM DTT, 1 mM PMSF) and lysed by sonication at ten seconds interval for 10 minutes and then centrifuged at 4 °C (12000 rpm, 30 min) to collect protein supernatant. The supernatant was filtered with a 0.45 μm filter and the filtrate was added into a Ni-NTA column (Bio-Rad) which had been equilibrated with buffer A before using. The Ni-NTA beads was set at 4 ℃ for 30min before being eluted with Buffer A containing 300 mM imidazole HT-SELEX was performed by using selection ligands containing  a 10 bp barcode after the 40 bp randomized region. The ligand libraries were produced by PCR amplification using the primers in Supplementary Table1 and 40 bp randomized oligo as template. The libraries were sequenced using BGI MGISEQ-2000 sequencer to analyze the uniformity of each base (A, T, C, G). RNA input was synthesized from the DNA-templates using T7 in vitro transcription and the remaining DNA template was digest by DNaseⅠ. To enable the RNA to form secondary structures, the RNA input libraries were then heated to 70°C followed by slow cooling. Then, 600 ng protein and 5 ul RNA input selection ligands were added into  binding buffer (50 mM NaCl, 1 mM MgCl2, 0.5 mM Na2EDTA , 1U/μL RNase inhibitor and 4% glycerol in 50 mM Tris-Cl, pH 7.5) until the total volume reached 25 ul. After incubated for 15 minutes at 37°C, the mixture was followed by additional 15 minutes at room temperature. Subsequently, 150- µL binding buffer, containing 10 µL pre-equilibrated Ni Sepharose 6 Fast Flow resin (GE Healthcare, 17-5318-01) was added into the mixture. After incubated for further 2 hours with gentle shaking at room temperature, the beads were then washed with gentle shaking 12 times with 200 μL of binding buffer to remove the unbound RNA ligands. Subsequently, the residual moisture on beads was carefully cleared by a soft centrifuging at 500 g for 30 s, and then the bound RNA was resuspended in 20 μL elution buffer (0.5 µM RT-primer, 1 mM EDTA and 0.1% Tween 20 in 10 mM Tris-Cl buffer, pH 7.0) and heated for 5 minutes at 70°C followed by 4°C to anneal the reverse transcription primer to the remaining RNA. Finally, the eluted RNA was used for reverse transcription and PCR amplification using the the primers in Supplementary Table 1, and the obtained PCR products were used as DNA template for the next cycle after containing a T7 promoter. This process was repeated four times. Each PCR products were purified and sequence using  BGI MGISEQ 2000 sequencer.