title: Illumina HiSeq 2000 paired end sequencing; The Nrd1-like protein Seb1 coordinates co-transcriptional 3’ end processing and polyadenylation site selection
design: The Nrd1-like protein Seb1 coordinates co-transcriptional 3’ end processing and polyadenylation site selection
library_construction_protocol: S. pombe cells were grown in EMM medium supplemented with 20 ug/ml Thiamine at 30C for 10 hours (final OD600: 0.5-0.6). Cells were harvested and fixed for 20 minutes with 1% formaldehyde (Larochelle et al. NAR 2012, 40:10240) ChIP experiments were performed as described in (Larochelle et al. NAR 2012, 40:10240). For ChIP-seq experiments, chromatin immunoprecipitation was done as described above with the following modifications. We scaled up the reactions to obtain larger amount of chromatin for library preparation. Chromatin was immunoprecipitated with Pan mouse IgG Dynabeads (Invitrogen) antibody against Rpb1 (8WG16, Convance). The 8WG16 antibody is known to recognize the C-terminal domain (CTD) of Rpb1 and measures total RNAPII along genes in yeast. Whole cell extract from fixed cells was prepared as described in (Larochelle et al. NAR 2012, 40:10240) Libraries were prepared using the NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina kit (New England BioLabs) according to the manufacturer instructions.