GSM1573455: POL_stat_phase (run37); Saccharomyces cerevisiae; OTHER
Per 5 OD600 units, cell pellets were resuspended in 200 µl lysis buffer (2% Triton X-100, 1% SDS, 0.5 M NaCl, 10 mM Tris-HCl pH8.0, 1 mM EDTA). An equal volume of TE-equilibrated phenol and glass beads (0.40-0.60 mm Φ, Sartorius) were added, and cells lysed by vortexing for 2 min; 200 µl of TE was then added, followed by an additional 1 min of vortexing. After centrifugation, the aqueous phase was further extracted with equal volumes of phenol:chloroform:isoamylalcohol (25:24:1) and chloroform. Total nucleic acids were precipitated with 1 ml of 100% ethanol, and dissolved in 0.5 M NaCl. RNA was degraded by treatment with 10 μg RNAse A (Roche) for 1 h at room temperature. DNA was finally purified with an equal volume of Ampure XP beads (Beckman Coulter) and eluted in nuclease-free water. DNA was sonicated using a Bioruptor Plus (Diagenode) to achieve an average fragment length of approximately 400 bp. Fragmented DNA was concentrated by ethanol precipitation and size selected using 1.2 volumes of Ampure XP. DNA was quantified by nanodrop (Thermo Scientific) and up to 5 μg was used for NEBNext End Repair and dA-Tailing (New England Biolabs) following the manufacturer’s guidelines. After the End Repair reaction DNA was purified using 1.2 volumes of Ampure XP. Subsequent steps were performed in the presence of Ampure XP beads, capturing the DNA by adding NaCl and PEG8,000 to final concentrations of 1.25 M and 10% respectively. The trP1 adapter (see below) was attached using NEBNext Quick Ligation with 120 pmol of adapter per μg of DNA for 14-18 h at 16°C. Terminal transferase (NEB) was then used to block any free 3’ ends with ddATP for 2 h at 37°C, with 20 U of TdT per μg of DNA. After Ampure XP purification, beads were removed and DNA nicked using recombinant RNase H2 (10 pmol per µg of library) or Nb.BtsI (NEB; 10 U per µg) for 2 h at 37°C. RNase H2 purification and reaction conditions were as previously described62. Enzymes were inactivated by heating at 80°C for 20 min, and DNA was purified using 1.8 volumes of Ampure XP. Shrimp Alkaline Phosphatase (Affymetrix; 5 U) was then used to remove 5’ phosphates at 37°C (1 h per μg of library). After heat inactivation for 15 min at 65°C and Ampure XP purification, DNA was denatured by heating at 95°C for 5 min and snap cooling. Subsequently, A adapter (see below; 120 pmol per μg of library) was attached using NEBNext Quick Ligation for 14-18 h at 16°C. Fragments with biotinylated A adapter were captured on streptavidin-coupled M-280 Dynabeads (Life Technologies) following the manufacturer’s guidelines, and non-biotinylated strands were released in 0.15 M NaOH. Single stranded fragments were concentrated by ethanol precipitation.
GEO
GSM1573455