nuclear run-on sequencing analysis of histone deacetylase inhibition
nuclear run-on sequencing analysis of histone deacetylase inhibition
MCF10A cells were cultured with DMEM/F12 media supplemented with 2.5% Horse serum, 0.5ug/mL hydrocortisone, 10ug/mL Insulin, 20ng/mL EGF and 100ng/mL cholera toxin. BT474, SKBR3 and ZR75-30 were cultured in 10% FBS containing RPMI 1640 at 37C with 5% CO2 When cells are 80% confluent, media was replaced and treated with either 500nM trichostatin A (TSA) or vehicle (DMSO) for 4 hours at 37C. We have followed the protocol previously published in Core, L., Waterfall, J. & Lis, J. "Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters." Science 322, 1845-1848 (2008). Briefly, nuclei from treated cells were isolated and incubated for 5 minutes with nucleotides and bromo-UTP analog. The nascent RNA from the labeling reaction was isolated and subjected to immunoaffinity purification using Br-dUTP antibody (Santa Cruz Biotech). After first round of purification of nascent RNA, the resulting RNA was ligated with 5' small RNA adaptor RNA (Illumina) and subjected to second Br-UTP purififcation. The resulting RNA is futher ligated using 3' adaptor RNA and subjected to third round of purification.  The final purified RNA was reverse transcribed and PCR amplified and size selected (150-250bp) using polyacrylamide gel electrophoresis to generate the final sequencing library.
Experimental Factor: COMPOUND1
trichostatin A
Experimental Factor: DOSE1
500 nM
Experimental Factor: COMPOUND2
cycloheximide
Experimental Factor: DOSE2
10 other
Experimental Factor: CELL_LINE
MCF10A