Genomics

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Genome-wide localization of the RNA polymerase III transcription machinery in human cells


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 “chromatin affinity purification” (ChAP) with streptavidin beads. In each case, the precipitated material was then processed for deep sequencing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE18184 | GEO | 2010/02/11

SECONDARY ACCESSION(S): PRJNA119545

REPOSITORIES: GEO

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