title: GSM8473006: MS023 1week + Dex 3hrs (29); Homo sapiens; RNA-Seq
library_construction_protocol: At the conclusion of the treatment, media was aspirated and cells were rinsed with 10mL of ice-cold PBS, scraped, and collected into 15mL conical tubes on ice. Cells were collected by centrifugation at 1,000xg at four degrees C for 5 minutes. PBS was aspirated and pellets were resuspended in 10mL ice-cold permeabilization buffer. Cells were incubated in permeabilization buffer for five minutes before being centrifuged at 1,000xg at four degrees for five minutes. Permeabilization buffer was aspirated and permeabilized cells were resuspended in 100uL freezing buffer and snap frozen before being stored at -80C. When preparing for the run on step, frozen permeabilized cells were thawed on ice. Run-on master mix was warmed to 37C and 100uL of 2x run-on master mix was added to 100uL of frozen cells before being placed in a 30C water bath for five minutes with slight agitation after 2.5 minutes of incubation. After five minutes of run-on, 500uL of Trizol LS was added and samples were vortexed until homogenized to stop the reaction. 130uL of chloroform was added, samples were vortexed for 15 seconds, and tubes were centrifuged at 15,000xg for five minutes at 4C. The aqueous phase was transferred to a new Eppendorf tube and RNA was precipitated using 2.5 volumes of 100% ethanol for 10 minutes before being centrifuged at 15,000xg for 20 minutes at 4C. The pellet was washed with 75% EtoH, centrifuged again, and ethanol removed completely before resuspending the RNA pellet in 19uL of DEPC-treated water. Sample quality was assessed by nanodrop and Tapestation before proceeding to RNA fragmentation with Ambion RNA fragmentation reagent (Ambion AM8740) for 4 minutes at 72C before adding stop solution to the reaction. Free NTPs were removed using a p30 column. Nascent RNA was enriched using streptavidin-coated Dynabeads (Thermo Fisher #65305), washed 2X with 500uL of ice cold high-salt wash buffer using a magnet. Beads were then washed 2X with 500uL binding buffer and 1X with low-salt wash buffer. Buffer was removed and beads were resuspended in 300uL of Trizol to dissociate nascent RNA from the beads. All buffers were made according to Mahat, 2016. 60uL of chloroform was added and vortexed.  The mixture was centrifuged at 15,000xg for five minutes at 4C. The aqueous phase was removed and moved to a separate tube before repeating the nascent RNA enrichment and wash steps. After the second round of purification, nascent RNA was precipitated with 1.25uL glycoblue and 2.5 volumes of 100% ethanol before centrifugation at 15,000xg for 20 minutes at 4C. Ethanol was removed and the RNA pellet was washed with 75% ethanol. RNA was centrifuged again  before removing the 75% ethanol completely. Pellets were resuspended in 5uL DEPC treated H2O and taken directly into library preparation using the NEBNext Ultradirectional II RNA sequencing kit (NEB #E7760S) following the manufacturer's instructions.