Transcription elongation analysis by GRO-seq
Transcription elongation analysis by GRO-seq
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. IMR90 cells were cultured in MEM with Earles salts with 10% FBS. H1 (from WiCell) embryonic stem cells were cultured on feeder-free matrigel coated plates in Dulbecco's modified Eagle medium (DMEM)/F12, supplemented with 1% MEM-nonessential amino acids (Invitrogen), 1 mM L-glutamine, 1% penicillin-streptomycin, 50 ng/mL basic fibroblast growth factor (Millipore), N2 supplements (1X), and B27 supplements (1X) (Invitrogen). 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: CELL_LINE
MCF 10A
Experimental Factor: CELL_TYPE
epithelial