GSM2190463: first tagged Seb1 CRAC sample; Schizosaccharomyces pombe; OTHER
Cells (0.8-1g) were lysed in 1V (w/v) of TN150 (50 mM Tris pH 8.0, 0.1% NP-40, 150 mM NaCl, 5 mM beta-mercaptoethanol, Roche protease inhibitor cocktail) and 3V of 0.5 mm Zirkonia beas by vortexing for five minutes at full speed. Lysates were clarified by centrifugation and processed as previously described (Granneman et al 2009, PNAS). Libraries were constructed using the CRAC library preparation method described in Granneman et al PNAS 2009 with small modifications to the adapter sequences (see sequences below) PCR oligonucleotides for making CRAC libraries 3' adapter: App_PE: 5’App-NAGATCGGAAGAGCACACGTCTG-ddC 3’ 5' adapters: L5Bb: 5'-invddT-ACACrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrNrNrNrCrArCrUrArGrCrN-OH-3' L5Bc: 5'-invddT-ACACrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrNrNrNrGrUrGrArGrCrN-OH-3' L5Cb: 5'-invddT-ACACrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrNrNrNrUrGrGrArGrCrN-OH-3' L5Cc: 5'-invddT-ACACrGrArCrGrCrUrCrUrUrCrCrGrArUrCrUrNrNrNrArCrUrCrArGrCrN-OH-3' P5_forward: 5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’ P3_reverse: 5’-CAAGCAGAAGACGGCATACGAGAT(6nt barcode)GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’
GEO Accession
GSM2190463