strain: FY11943 genotype: 3xFlag:ago1-KanMX6 ars1::pREP1GFP-HP-LEU2 arg3::ura4- GFP arg11::HphMX6 his3-D1 leu1-32 ura4-D18 ade6-210/216 purification: FLAG Immunoprecipitation molecule: small RNA
Growth protocol
Strains were grown in PMG complete to keep the GFP hairpin, which is under the nmt1 promoter, expressed. Standard procedures were used for growth and genetic manipulations (Moreno et al., 1991).
Extracted molecule
total RNA
Extraction protocol
Total small RNA was obtained by resuspending cells in RNA extraction buffer (50 mM Tris–HCl pH 7.5, 10 mM EDTA pH 8, 100 mM NaCl, 1% SDS), lysed with phenol:chloroform 5:1 and acid washed beads. The soluble fraction was extracted with phenol/chloroform. Small RNA were concentrated by precipitating away large RNAs with 10% polyethylene glycol 8000, 0.5 M NaCl and then by ethanol precipitation. For gel-purified small RNA, 10 to 30-nt siRNAs, from 400 μg of total small RNAs, were excised from a 12% polyacrylamide gel. After elution from the gel (incubation over-night in RNA extraction buffer at 4°), small RNAs were extracted with phenol/chloroform was and precipitated with ethanol, sodium acetate, and glycogen. For Ago1-associated small RNA preparation 10 g of cells were lysed in presence of liquid nitrogen using a mortar grinder (Retsch) for 30 minutes. Extracts were prepared by dilution of the crushed cells in 20 ml lysis buffer (50 mM Hepes-NaOH (pH 7.5), 150 mM NaCl, 1 mM MgCl2, 0.1% NP-40, 5mM DTT, 1x Roche EDTA-free protease inhibitors cocktail, 0.5mM PMSF, 1/100 super RNAsin (Ambion), and filtration through GD/X 1.6 μm (Whatman). Immunoprecipitations were performed using 20 μg of M2 anti-Flag antibody (Sigma F1804) coupled to 4 μL protein G Dynabeads resin (Dynal) for 15 minutes at 4 °C. RNA samples bound to Dynabeads were washed once with lysis buffer and once with lysis buffer with 2 mM MgCl2, treated with 200 ng/ml proteinase K (Sigma), extracted with phenol/chloroform and chloroform. RNA was precipitated with ethanol, sodium acetate, and glycogen. Libraries were prepared of the small RNA according to Illumina's instructions for subsequent cDNA sequencing.
Library strategy
RIP-Seq
Library source
transcriptomic
Library selection
other
Instrument model
Illumina Genome Analyzer
Data processing
Normalised wiggle files were created by measuring the number of reads that mapped to each base. Each read counted 1/n towards each base that it align to, where 'n' is the number of times a read could be aligned in the genome. .wig files are give for each strand and both strands combined for the wild type and rpd1delta only.
Two additional constructs were mapped, ura4GFP (ura4 gene combined with gfp gene) and GFPHP (two gfp genes that form a hairpin). A gff file describing these constructs and the fasta files for these contructs are included as supplementary files linked to GSE20330.
