GSM7951752: 2tj1E polyA RNA; Schizosaccharomyces pombe; RNA-Seq
ChIP libraries: IP of crosslinked (1% formaldehyde, 15 min) sonycated lysate, followed by decrosslinking and protein and RNA degradation. DNA library construction with NEBNext Ultra II DNA Library Prep Kit for Illumina kit (NEB). DNA sequencing via Illumina: Cells were lysed by bead beating and lysate was sonycated and RNAse-treated. DNA isolated via phenol-chloroform-isoamylalcohol purification followed by ethanol precipitation. DNA library construction with NEBNext Ultra II DNA Library Prep Kit for Illumina kit (NEB). DNA sequencing via Nanopore: DNA extraction was performed with Genomic Tip 500/G kit (Qiagen) following the manufacturer's instruction, but using Lallzyme MMX for lysis. Sequencing was performed using Oxford Nanopore (MinION). RNA sequencing: RNA was isolated applying the hot phenol method. Total RNA and polyA RNA Libraries made with NEBnext Ultra Directional RNA Library Prep Kit for Illumina (NEB). In the polyA library preparation, isolation of the polyA RNA from the total RNA was performed using polyT beads. Small RNA sequencing: strains with the endogenous Ago1 3xFLAG tagged were grown in YES and 2.5l of log phase cells were collected. Cells were resuspended in lysis buffer (50 mM HEPES pH 7.5, 1.5 M NaOAc, 5 mM MgCl2, 2 mM EDTA pH 8, 2 mM EGTA pH 8, 0.1% Nonidet P-40, 20% glycerol) supplemented with 1mM PMSF and Complete EDTA free Protease Inhibitor Cocktail (Roche), keeping a 1:1 ratio between the cell volume and the buffer. Cells were fragmented using glass beads and the bead beater as described for the ChIP protocol. The cell debris was eliminated by cetrifugation at 13000g and the crude lysate was incubated with 30μl of FLAG-M2 agarose resin (Sigma-Aldrich A2220) at 4°C for 2h with gentle rotation. The IP was washed five times with the lysis buffer and Ago1 was eluted from the resin mixing 1% SDS with 300 mM NaOAc and phenol-chloroform-isoamylalcohol (25:24:1, Roth). The sRNAs were concetrated by ethanol precipitation, run on an 18% polyacrylamide urea gel together with 22, 26 and 30 nt RNA markers and the bands of sizes between 20 and 30 nt were excised from the gel. A 3' linker was ligated to the sRNAs (5 rAppCTGTAGGCACCATCAAT–N ′ H2 3', NEB, reaction concentration 2μM). The 3' ligated products were purified on an 18% acrylamide urea gel with subsequent phenol-chloroform purification and ethanol purification. The 5' adaptor (5'-ACACUCUUUCCCUACACGACGCUC UUCCGAUCU-3', Metabion) ligation was performed, followed by reverse transcription (RT primer: 5'- GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCGATTGATGGTGCCTACAG -3', Metabion). The cDNA was amplified with 12-20 PCR cycles using the Phusion hot start II DNA polymerase kit (ThermoFisher) and the Illumina P5 5' primer (5' -AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC G -3') together with the Illumina P7 3' primer with inserted barcode (5'-CAA GCA GAA GAC GGC ATA CGA GAT XXXXXX GTG ACT GGA GTT CAG ACG TG -3'). The sRNA library was purified from an 8% polyacrylamide gel, ethanol precipitated and Single end sequencing was performed on an Illumina GAIIX sequencer. Libraries were prepared using NEBnext Ultra Directional RNA or DNA Ultra II Library Prep Kit for Illumina (NEB), following the manufacturer instruction.