title: GSM5259588: In-line-seq; synthetic construct; OTHER
library_construction_protocol: Quenched RNA samples were purified with 2 µL of Oligo dT bead (Thermo Fisher, AM1922) and 0.8 µL of 10 µM FAM-A20-Tail2. RNA, bead, and primer were incubated at room temperature for 15 min, pulled down by magnetic stand for 10 min, and washed twice with 70% ethanol and left to dry on the bead. RNA was eluted in 2.5 µL of nuclease-free water (RNA was binded with OligodT bead and FAM.A20 Tail2 at this point) 5 µL of purified RNA was added to a reaction mixture containing 1x First Strand buffer (Thermo Fisher), 5 mM dithiothreitol (DTT), 0.8 mM dNTPs, 0.6 µL of SuperScript III RTase (Thermo Fisher) to a final volume of 15 µL. The reaction was incubated at 48 °C for 40 minutes and stopped with 5 µL of 0.4 M sodium hydroxide. The reaction was then incubated at 90 C for 3 minutes, cooled on ice for 3 minutes, and neutralized with 2 µL of quench mix (prepared as 2 mL of 5 M sodium chloride, 3 mL of 3 M sodium acetate, 2 mL of 2 M hydrochloric acid). cDNA was pooled down with 1.5 uL of Oligo C' beads64 (in house magnetic beads prepared by immobilizing 2x Biotin oligonucleotides with Dynabeads™ MyOne™ Streptavidin C1; Thermo Fisher Scientific 65001), washed twice with 70% ethanol, then resuspended in 3.0 µL of water. We pooled 1.5 µL of each sample together, and took 9 µL of cDNA to continue to ligation an Illumina adapter by using Circ. Ligase I (Lucigen) at 68°C for 2 h. The reaction was stopped by incubation at 80 °C for 10 min. cDNA was added to 10 ul of 5 M NaCl and pulled down with a magnetic stand and washed with 70% ethanol; the ligated product was resuspended in 15 µL H2O.