title: GSM3184653: IP_Rpb1_mut-ysh1_MM+rapamycin_SI_exp5; Schizosaccharomyces pombe; Saccharomyces cerevisiae; ChIP-Seq
library_construction_protocol: ChIP-seq experiments of total RNAPII and phospho-CTD modifications in wild-type and dhp1-depleted cells were performed with a spike-in adjustment procedure that allows a quantitative comparison between independent strains or conditions (Orlando et al. 2014). Chromatin preparations of S. pombe FBY1995 (Rpb3-HA; WT) and FBY2016 (Rpb3-HA; Pnmt-dhp1) strains and S. cerevisiae FBY2064 strain (reference strain containing a copy of HA-tagged histone H2B) were done as a regular ChIP, except that chromatin extracts have been frozen in liquid nitrogen and stored at -80°C before affinity purifications. For Tyr1-P, Ser2-P, Ser5-P, Ser7-P, and 8WG16 IPs, a S. pombe/S. cerevisiae chromatin ratio of 0.9:0.1 was used, whereas a chromatin ratio of 0.995:0.005 was used for Rpb3/H2B-HA purifications. Following combination of S. pombe/S. cerevisiae chromatin extracts, ChIP assays were performed as described above using antibody-coated beads. ChIP-seq profiles of total RNAPII from WT and ysh1-AA strains were also performed with a spike-in adjustment (ratio S. pombe/S. cerevisiae of 0.9:0.1) using extracts from S. pombe that were previously grown in EMM leu- and treated for 4 hours with 2.5 μg/ml of rapamycin. DNA librairies for ChIP-seq experiments were prepared as described previously (Lemay et al. 2016) using either the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina kit (New England BioLabs) or the SPARK DNA Sample Prep Kit Illumina Platform (Enzymatics) according to the manufacturer's instructions.