| Construction Protocol | We used the recombineering technology (Liu, P., Jenkins, N.A. and Copeland, N.G. (2003) Genome Res.) to generate the targeting vectors that introduce the triple affinity tag in the subunits of Pol III, TFIIIB, TFIIIC, and TFIIS. The 46C ES cell line (Ying, Q.L., Stavridis, M., Griffiths, D., Li, M. and Smith, A. (2003) Nat. Biotech.) was transfected by electroporation with each targeting vector. Cells were plated in D15 medium as described (Tessarollo, L. (2001). Methods Mol Biol.) and selected with G418. Individual ES cell colonies were collected seven days after electroporation, amplified and genotyped by Southern blotting, in order to identify the clones that underwent a homologous recombination event. In these clones, a sequence encoding a 6 Histidine-Flag-HA tag followed by a neomycin resistance marker flanked by loxP sites, was inserted just after the last codon of the gene encoding the protein to be tagged. For TCEA1, the tag was inserted just after the start codon. The integration of the cassette at the right loci was verified by Southern blotting using three or four different restriction enzymes and PCR. Transient transfection with a Cre recombinase expression vector was used to remove the selection cassette. The karyotypes of all cell lines were verified. The genes that were modified in the cell lines encoded RPC1 (MGI:2681836), RPC4 (MGI:1914315), BRF1 (MGI:1919558), BRF2 (MGI:1913903), TFIIIC220 (MGI:107887), TFIIIC110 (MGI:1919002), TFIIIC90 (MGI:2138937) and TCEA1 (MGI:1196624) respectively. The expression of the tagged versions of the proteins was verified by western blotting using HA7 anti-HA antibodies (Sigma). Untagged ES cell lines (46C, 46C-2) are used as a negative control for the ChIP-seq experiment. ES cells were cultured on embryonic fibroblast feeder cells blocked with mitomycin C in D15 medium (Dulbecco Modified Eagle Medium High Glucose supplemented with 15% FBS, 2mML-glutamine, 50 units/ml penicillin, 50 ug/ml streptomycin, 0.1 mM nonessential amino acids (all from GIBCO), 0.1 mM 2-mercaptoethanol [Sigma] and 1,000 units/ml LIF). ES cells were maintained at 37C, 5% carbon dioxide, fed with fresh media daily, and transferred to new plates after trypsinization. Typically 150-200 million cells were collected for each ChIP experiment. The cellular proteins and DNA were cross-linked by the addition of formaldehyde (1% final concentration) in cell culture media. The plates were incubated for 10 min at room temperature then, the reaction was stopped by the addition of glycine (0.125 M final concentration). The cells were washed twice with 10 ml chilled PBS. Each plate was scraped with 2 ml PBS with protease inhibitors (Roche complete, 10 mM PMSF dissolved in ethanol). Untagged ES cells (46C-2), TFIIIC subunits and TCEA1 ChIPs were fragmented using MNase I. The protocol will be described in details elsewhere (M. Gerard, manuscript in preparation). |
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