title: GSM1467865: Wild type; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: 1.6x10^10 sporulating cells were harvested and washed with PBS. The cells were resuspended in one pellet volume of lysis buffer (50mM HEPES pH7.5, 50mM KCl, 5mM MgCl2, 1mM DTT, cOmplete Mini EDTA-free Protease Inhibitors) and frozen drop-wise on liquid nitrogen. Cells were ground to a fine powder in a pestle and mortar under liquid nitrogen and thawed in a 50 ml tube on ice. The lysate was centrifuged for 5 min at 4500 rpm and the supernatant clarified by centrifugation for 20 min at 30,000g. Cleared lysate was transferred to a 15 ml tube and incubated with 100µl of IgG sepharose beads (G.E. Healthcare) for 2 hours at 4degC with gentle agitation. Beads were washed five times for 10 minutes at 4degC with IP Buffer (10mM Tris pH7.5, 120mM NaCl, 5mM MgCl2, 0.1% NP-40, 1mM DTT). Beads were incubated for 2 hours at 16degC with 100µl IP buffer and 1µl AcTEV protease (Invitrogen). Beads were removed by centrifuging through a Micro Bio-Spin column (Bio-rad), and the flow-through was diluted with 500µl IP buffer + 2mM CaCl2 and incubated for 50min at 4degC with 100µl calmodulin sepharose beads (G.E. Healthcare). Beads were washed three times for five minutes with IP buffer + 2mM CaCl2 and protein was eluted in 100µl calmodulin elution buffer (10mM Tris pH7.5, 120mM NaCl, 5mM EGTA). Beads were removed by centrifuging through a Micro Bio-Spin Column, and RNA was extracted by phenol:chloroform extraction and ethanol precipitation. RNA was fragmented in 200mM Tris acetate pH 8.2, 500mM KOAc, 150mM MgOAc2 for 3 min at 94deg, ethanol precipitated and reverse transcribed from random hexamers using Superscript II (Life Technologies) according to manufacturer’s instructions. Tris pH7.8 and MgCl2 were added to 50mM and 5mM respectively, and second strand synthesis was performed with 2U RNase H (NEB) and 50U DNA polymerase I (NEB) at 16deg for 2.5 hours. Ends were repaired using 15U T4 DNA polymerase (NEB), 5U Klenow DNA polymerase (NEB) and 50U T4 PNK (NEB) in T4 DNA ligase buffer (Life Technologies) at 20deg for 30 min. cDNA was tailed with dATP using 15U Klenow (3’-5’ exo-) (NEB) in NEBuffer 2 at 37deg for 30 min, and 1:10 diluted Illumina adapters were ligated using a Ligafast kit (Promega) followed by gel purification of ~200bp species. Libraries were amplified for 15 cycles using Phusion polymerase (NEB) and Illumina PCR primers 1.0 and 2.0.