As for ChIP with protease inhibitors (mini-complete EDTA free tablets, Roche Applied Science, Welwyn, UK). Cells were broken using glass beads and a Fastprep machine (20 seconds at 6.0 m/s) and then sonicated using a Bioruptor (Diagenode, Li e, Belgium) with 6 minutes total time (15 s on, 30 s off). DNA was phenol/chloroform extracted twice, and the resulting material was RNAse treated for 20 minutes before re-precipitating. The resulting DNA was then labeled according to standard Affymetrix protocols. The log 2 FAIRE signals were normalized by subtracting the average PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/24735385 signal of three genomic DNA hybridizations to correct for GC bias.Probe mapping for bulk signal differencesmultiple times, the last ten bins of every upstream exon, the ten bins of every intron, and Dasotraline the first ten bins of every downstream exon were used PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26205627 to average probe signals from the various experiments. Probes falling in the first ten bins of every first exon and the last ten bins of every last exon were averaged to create the first and last ten bins for upstream and downstream exons, respectively.Average gene calculations by expression groupReplicate gene expression data collected previously [7] from Affymetrix Yeast 2.0 Genechip?arrays were first filtered for undetectable signal (< 1; 480 of 5,296 genes excluded) and then sorted into spliced and unspliced genes (2,218 and 2,598 genes, respectively). Lists of spliced/unspliced genes were then ranked in descending order and split into 10 equal groups (approximately 220 and 260 genes per group for spliced and unspliced genes, respectively). Average gene profile calculations were then performed as described above for genes within each expression bin.Accession numbersAll microarray data used have been submitted to ArrayExpress under the accession number E-TABM-946.For analyzing differences in Pol II, histone H3 and H3K36me3 ChIP or FAIRE signals in introns and exons, 25-bp Affymetrix probes were mapped back to the S. pombe genome (GeneDB). Probes where the entire 25 bp length fell within an intron or exon were classed as `intron probes' or `exon probes', respectively. All probes that fell entirely within the ORF of unspliced genes were used for calculating the signal of unspliced genes.Average gene calculationsAdditional materialAdditional file 1: Single-gene examples of Pol II occupancy. (a-f) Affymetrix tiling array data for RNA Pol II is shown for three genes (SPAC13G7.11 (a), SPBC1773.01 (b), SPCC126.05c (c)) with low expression (ranked 2,447,1,666, and 2,310 out of 4,816, respectively, according to Affymetrix expression data) and three genes (SPBC4F6.18c (d), SPAC17G6.06 (e), SPCC24B10.09 (f)) with high expression (ranked 216, 90, and 56 out of 4,816, respectively, from data as above). Additional annotated features are shown (expression rankings are SPAC13G7.12c (3,307), SPBC1773.02c (2,764), SPCC126.04c (2,123), SPCC126.06 (2,591), SPBC4F6.17c (1,944), SPAC17G6.05c (4,227), SPAC17G6.07c (811), SPCC24B10.08c (3,300), SPCC24B10.10c (3,376)) and the range of absolute values of RNA Pol II signals (as previously calculated [7]) are shown on the left side of each panel. Introns within genes shown are indicated by red lines. Additional file 2: Expression level of spliced genes is not biased by intron size. A scatterplot showing the size of each intron in the annotated S. pombe genome and the corresponding gene expression level (according to previously published Affymetrix microarray data [7]). Additional file 3: Validation.