Genome-wide localization of the RNA polymerase III transcription machinery in human cells
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ABSTRACT: To characterize the pol III transcriptome in actively growing human cells, we used MR90hTeIrt lung fibroblasts, which are immortal but not transformed, with an intact Rb pathway. After formaldehyde cross-linking, chromatin was extracted and submitted to immunoprecipitation. To identify actively transcribed RNAP-III transcription units, we used antibodies directed against the RPC4 subunit of RNAP-III. To identify promoter regions, we used antibodies directed against Bdp1, which is part of both Brf1-TFIIIB and Brf2-TFIIIB, and thus should mark all types of active RNAP-III promoters. Antibodies directed against Brf1 were used to mark type 1 and 2 promoters. For type 3 promoters, we engineered an IMR90hTert cell line expressing the SNAPc subunit SNAP45 tagged at its C-terminal end with the biotin acceptor domain as well as the biotin ligase BirA and performed M-bM-^@M-^\chromatin affinity purificationM-bM-^@M-^] (ChAP) with streptavidin beads. In each case, the precipitated material was then processed for deep sequencing. ChIP-enriched DNA or ChAP-enriched DNA from MR90hTeIrt cells were analyzed by Illumina high-throughput sequencing. This analysis includes ChIP data done with antibodies against : RPC4, Bfr1, Bdp1 and SNAP45. Two lanes were run for RPC4, Brf1 and SNAP45, and three for Bdp1. One lane was run with Input DNA for control.
ORGANISM(S): Homo sapiens
SUBMITTER: Viviane Praz
PROVIDER: E-GEOD-18184 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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