Make Your Life A Lot Easier Thanks to Non-specific serinethreonine protein kinase Understanding

Serial 10-fold dilutions of the strains indicated were plated on PMGc (nonselective, NS) or on PMGc plates containing 2 mg/ml 5-FOA. Chromatin immunoprecipitation was performed as described in Buhler et?al. (2006). An antibody against dimethylated H3-K9 (abcam, #ab1220) was used. Centromeric siRNAs were detected by northern blotting as described in Buhler et?al. (2006). RNA was isolated from cells collected at OD600 = 0.5 using the hot phenol method (Leeds et?al., see more 1991). The isolated RNA was processed according to the GeneChip Whole Transcript (WT) Double-Stranded Target Assay Manual from Affymetrix using the GeneChip S. pombe Tiling 1.0FR. For analysis of the tiling arrays, an R-based script was used, which is available upon request. We used the genome and annotations from the S. pombe Genome project (ftp://ftp.sanger.ac.uk/pub/yeast/pombe/GFF/pombe_160708.fasta;ftp://ftp.sanger.ac.uk/pub/yeast/pombe/GFF/pombe_160708.gff). The oligos from the Affymetrix selleck chemicals .BPMAP file were remapped using bowtie, and the .GFF file was used to map them to the genes. The resulting .CDF file is available upon request. Total RNA was isolated from cells harvested at OD600 = 0.5 using the hot phenol method (Leeds et?al., 1991) and subjected to size fractionation using RNeasy Midi columns (QIAGEN) as previously described (Buhler et?al., 2006). 17�C30 nt small RNAs were PAGE purified and were cloned based upon the preactivated, adenylated linkering method described previously (Lau et?al., 2001) using a mutant T4 RNA ligase (Rnl21-249) (Ho et?al., 2004). All samples were barcoded at the 3�� end of the 5�� adaptor using a hamming distance two code with a 3�� cytosine (AAAC, ACCC, AGGC, ATTC, CACC, CCGC, CGTC, CTAC, GAGC, GCTC, GGAC, GTCC, TATC, TCAC, TGCC, TTGC) and sequenced in one lane of an Illumina GA2 instrument. Individual reads were assigned to their sample based on the first four nucleotides containing the barcode. The 3�� adaptor was removed by aligning it to the read allowing one or two mismatches in prefix alignments of at least seven or ten bases, respectively. Low-complexity reads were filtered out based on their dinucleotide entropy (removing Non-specific serine/threonine protein kinase All the reads that were shorter than 14 nucleotides were removed. Alignments to the S. pombe genome (May 8, 2009, http://www.sanger.ac.uk/Projects/S_pombe/) were performed by the software bowtie (version 0.9.9.1) ( Langmead et?al., 2009) with parameters -v 2 -a -m 100, tracking up to 100 best alignment positions per query and allowing at most two mismatches. To track genomically untemplated hits (e.g., exon-exon junctions or missing parts in the current assembly), the reads were also mapped to an annotation database containing known sequences (http://www.sanger.ac.uk/Projects/S_pombe/). In that case, all best hits with at most two mismatches were tracked. Each alignment was weighted by the inverse of the number of hits.