GSM9154483: wt_rep2; Schizosaccharomyces pombe; RNA-Seq
ChIP-seq: Briefly, S. pombe cell cultures were grown in YES to an O.D600 of 0.5 – 0.6 and crosslinked with 1% formaldehyde for 15 minutes at room temperature. Crosslinking was terminated by adding 2.5 M glycine to a final concentration of 125 mM for 5 minutes. Cells were pelleted by centrifugation, washed with cold 1x TBS, frozen on dry ice, and stored at -80°C. Cell pellets from 50 ml cultures were resuspended in ChIP lysis buffer I and lysed in a Mini-Beadbeater-16 in the presence of glass beads (0.1 mm diameter) at 4°C for 1 minute (with 1 minute intervals on ice). Lysates were centrifuged at 16,100 x g for 20 minutes at 4°C. Pellets containing chromatin were resuspended in 1 ml ChIP lysis buffer II and transferred to 15 ml polycarbonate tubes. Lysates were sonicated for 30 minutes at 4°C (30 seconds ON, 30 seconds OFF, output setting high) using a water bath sonicator, transferred to new 1.5 ml tubes, and centrifuged at 16,100 x g for 20 minutes at 4°C. 250 – 300 µg of chromatin extract was incubated with antibodies. Extracts from S. cerevisiae strain BY4742 were also prepared in parallel and spiked into each immunoprecipitation (1% of the weight) for normalization. RNA-seq: S. pombe cultures were grown to an OD₆₀₀ of ~0.5. Cells were harvested by centrifugation, flash-frozen on dry ice, and stored at –80°C. Pellets were resuspended in 1 mL QIAzol and lysed using a Mini-Beadbeater-16 with glass beads at 4°C for 1 minute per cycle (with 1-minute intervals on ice) for a total of 5 cycles. Total RNA was extracted using the miRNeasy Micro Kit (Qiagen #1071023), following the manufacturer's instructions. RNA quantity and purity were assessed using a NanoDrop 2000 spectrophotometer. One microgram (1 μg) of total RNA was reverse-transcribed into cDNA using the SuperScript III First-Strand Synthesis Kit (Invitrogen #18080-051) with random hexamer primers ChIP-seq: Preparation of multiplexed ChIP-seq libraries from purified immunoprecipitated chromatin and sequencing were performed as described. In brief, the NEBNext Ultra II DNA Library Preparation kit (E7103S) was used to generate libraries using 5-10 ng of input or immunoprecipitated DNA and barcode adaptors (NEBNext Multiplex Oligos for Illumina (Set 1, E7335 and Set 2, E7500). RNA-seq: 1 μg of total RNA from each sample was used to prepare strand-specific libraries using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760S), according to the manufacturer's protocol. After poly(A) selection and fragmentation, first and second strand cDNA synthesis was performed. The resulting double-stranded cDNA was purified using SPRIselect beads (Beckman Coulter #B23317). Adaptors were diluted 5-fold and used for ligation, followed by size selection (~200 nt) using SPRIselect beads. Libraries were enriched by PCR amplification (10 cycles) using NEBNext Ultra II Q5 Master Mix, Universal PCR Primer, and Index (i7) Primers (NEB #E7335S). Final libraries were purified with SPRIselect beads, and their concentration and quality were assessed using Qubit Fluorometric Quantitation and the Bioanalyzer High Sensitivity DNA Kit, respectively.