GSM7735142: 5 MCF7 Fulvestrant 15min; Homo sapiens; OTHER
Run-on and subsequent PRO-seq library preparation experiments were performed on permeabilized cell pellets in a freeze buffer as described, but with modifications, from the Mahat 2016 Protol. Briefly, permeabilized cells were mixed with run-on buffer and incubated at 30°C for 5 minutes. Run-on reaction was stopped and RNA was extracted. RNA was fragmented. RNA was cleaned up using columns and reconstituted to a final volume. Biotinylated RNAs were enriched using the streptavidin beads. Binding and incubation occured. Bound beads were washed with a high salt buffer, twice with binding buffer, and once with low salt buffer. RNA was removed from the streptavidin beads. Extracted RNA was dissolved and subjected to a second round of biotinylated RNA enrichment. After the second round, the isolated RNA was dissolved and immediately proceeded to cDNA synthesis. The RNA libraries were prepared and sequenced using the Illumina NextSeq500, single read, 1x75 bps