GSM5169138: A549_WT_100nMdex; Homo sapiens; OTHER
Cells were kept on ice during the extraction protocol. Cells were washed with PBS, then incubated in PBS supplemented with 10 mM PBS for 5 minutes. Cells were scraped and placed in 15 mL Falcon tubes, then washed twice with PBS. Cells were then incubated in permeabilization buffer for 5 minutes, pelleted, and washed twice with permeabilization buffer. Cells were resuspended in freezing buffer and flash frozen. Run-on reactions with biotin-11-CTP and biotin-11-UTP were performed for 5 minutes. RNA was isolated, fragmented on ice with 0.2 N NaOH, and underwent buffer exchange with a P-30 column. Three biotin enrichments were performed with Dynabead streptavidin beads, and between enrichments, the 3' adapter was ligated, the 5' end was repaired with RppH and PNK enzymatic treatments, and the 5' adapter was ligated. RT was performed with SuperScript III, and 13 cycles of PCR were used to amplify the library, followed by clean-up with Ampure beads (1.6x ratio).
GEO
GSM5169138