GSM7890223: Ribo-WT_Rep1; Homo sapiens; OTHER
Cells were lysed in 5 µl of lysis buffer containing 20 mM BisTris (titrated, pH 7.2), 1.0% Triton-X100, 5 mM MgCl2, 5 mM CaCl2, 100 mM NaCl, 100 µg/mL cycloheximide, and 1 mM DTT using a pipette. Digestion was initiated by adding 1 µl of RNase I (NEB) at a dilution of 1:300, followed by incubation at 37°C in a thermal cycler with a heated lid for 15 minutes. The reaction was terminated by adding 1 µl of 1% SDS. Subsequently, samples were promptly placed on ice and kept there until processing through Ribo-ITP. Ribo-ITP and library preparation were conducted following the method described in the cited paper (Ozadam, H. et al, 2023, Nature). Libraries were generated using the D-Plex Small RNA-seq kit (C05030001, Diagenode) with minor modifications as outlined in the paper. Each sample was processed using a unique dual index module, and the complementary DNA was amplified for 12 cycles.