EDC-mediated attachment of poly(A)site-cleaved RNA to RNA polymerase II. (A) The plasmid construct and the outline of the experimental protocol (Rigo et al. 2005). (B) Pull-down of poly(A)site-cleaved RNA (Method 1). The DNA oligo for RNaseH cutting was CCATCTTCTGCCAGG (5′ → 3′) at 1 ng/μL. The concentration of this oligo was chosen so that only a small proportion of the transcripts would be cut by RNaseH at this upstream location. For lanes 1 and 2 the polymerase antibody used was N-20, lot L1407. Lanes 7 and 8 are from a different experiment using antibody from lot I0706. For lanes 1, 2, 5, and 6 the average and standard deviation are given for the experiment shown plus two additional independent experiments. For lanes 3 and 4 the average and range are given, for this and one additional independent experiment. For lanes 7 and 8 the average and range are for duplicates within the same experiment. The lane 7 and 8 data are not included in the average for lanes 1 and 2. (C) The amount of RNAPII and CstF pulled down concomitant with poly(A)site-cleaved RNA. The experiment was carried out as for part B except that each reaction was scaled up by twofold and then split in half for the RNA or the Western analysis. For simplicity, only the poly(A)site-cleaved RNA and the relevant protein bands are shown.
