GSM9226154: NT_TS_RIP; Homo sapiens; RIP-Seq
Cells were harvested, counted, and resuspended in cytoplasmic extraction buffer then centrifuged; the pellet was extracted with nuclear extraction buffer. Cytoplasmic and nuclear extracts were combined, and glycerol was added. Lysates were pre-cleared with magnetic Protein A/G beads, then subjected to RIP with anti-TS antibody (Proteintech 15047-1-AP) and captured on Protein A/G beads. Beads were washed, eluted in elution buffer (also containing proteinase K) at 60 °C for 30 min, and RNA was purified using the Geneaid VR050 kit. RNA concentration was measured by Qubit and integrity checked by agarose gelelectrophoresis. The libraries for Illumina sequencing were prepared using xGen RNA Lib Prep Kit (Integrated DNA Technologies, Iowa, USA) according to the Manufacturer's suggestions.