normalization data transformation protocol
Paired-end reads of length 51 bases (each part) originating from each sample were aligned using Bowtie 0.12.7 to the S. pombe genome sequence (Ensembl S. pombe, Build EF1) as well as to its corresponding exon-exon junctions database (only thesecond part of the paired-end reads was considered). Up to 3 mismatches were allowed. Reads that matched multiple loci were removed from further analysis and the resultant alignment files were processed to generate ‘pile-ups’ against each chromosome. Searches were performed against the genome sequence combined with a dataset of known exon-exon junctions as defined by Ensembl S. pombe release-13. To ensure that a 51-base read mapped to a splice junction, only the last 45 bases of the first exon and the first 45 bases of the second exon were considered (if the exon length exceeded 45 nucleotides). In this way, reads that overlapped a junction by less than 6 nucleotides were excluded. Reads that matched to more than one junction or elsewhere in the genome were also discarded. Genome_build: Ensembl S. pombe, Build EF1, version 58.1a Supplementary_files_format_and_content: Expression table and RPKM summary for all samples tab-delimited
Two biological replicates of the WT, Y1F, S2A, T4A, S7A,Y1F-S7A, S2A-S7A and T4A-S7A cultures were grown in YES (yeast extracts plus supplements) media at 30°C till they reached OD600 0.4 -0.7.
nucleic acid library construction protocol
Cells were harvested and RNA was isolated using the Qiagen RNeasy kit. RNA quality was assessed on a Bioanalyzer instrument (Agilent) and 1µg of total RNA was used to prepare polyA+ RNA using the Illumina TruSeq RNA sample kit . RNA-seq libraries were prepared from polyA+ RNA using the Illumina TruSeq stranded total RNA Sample Prep Kit according to the manufacturers protocol. Indexed libraries were normalized and pooled (8 samples per lane) for paired-end sequencing performed using an Illumina HiSeq 2000 instrument at the Weill Cornell Medical College Genome Core Facility (in New York).