GSM1654366: S. pombe WT; Schizosaccharomyces pombe; OTHER
Pellets were resuspended in in 1mg/mL zymolyase 100T in CPS buffer (50mM citrate phosphate buffer, pH=5.6, 1.2M sorbitol), treated for 1 hr at 37°C (or until >90% have lost their refringence under brightfield microscopy), then spun down for 3’ at 3,000g and resuspended in 5xTE (50mM Tris-HCl, 5mM EDTA, pH=8). 1/10th volume of 10% SDS was added and cells were lysed by incubation at 65°C for 5 minutes. Next, KAc was added to 1.25M final and incubated 30’ on ice. Samples were then spun to remove cell debris and ethanol precipitated. DNA pellets were then brought up in TE + RNase A (100μg/mL) and treated for 1 hour at 42°C. Supernatant was extracted with phenol/chloroform/iso-amyl alcohol (pH=8) twice, then concentrated by ethanol precipitated a final time. Pellets were then washed with 70% ethanol once before being resuspended in a small volume of TE or EB (10mM Tris-HCl, pH=8). After DNA purification, samples were digested with MseI to completion, purified and ligated to the oM536/oM537 linker. Libraries were prepared for high-throughput sequencing by PCR with oM538/544 (for WT), and oM538/543 (for sap1-c).
GEO Accession
GSM1654366