title: Illumina MiSeq 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). 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. Total RNA was isolated from S. pombe cells using a hot phenol method followed by phenol-chloroform extractions, and precipitation as described (Lyne et al., 2003, BMC Genomics 4, 27.). 10ug total RNA was used as starting material for strand-specific RNA-seq library preparation using an unreleased early version provided by Illumina of the Illumina TruSeq Small RNA Sample Prep Kit. Briefly: poly(A)+ RNA was enriched by two rounds of poly(dT) Sera-Mag magnetic beads purification. Enriched poly(A)+ RNA was then fragmented to an average size of ~200nt. Fragmented RNA was 3' de-phosphorylated with Antartic phosphatase and 5' phosphorylated with polynucleotide kinase this treatment prepares RNA fragments for subsequent ligation of Illumina RNA adaptors to their 5' and 3' ends using a 3' RNA ligase and a T4 RNA ligase, respectively. First strand cDNA was produced using a primer specific for the Illumina 3' adaptor. The library was finally amplified with 15 cycles of PCR using primers specific for the Illumina adaptors and purified using SPRI-beads (Agencourt, Beckman Coulter). Libraries size distributions and concentrations were determined on an Agilent Bioanalyzer