alias: GSM4546574
title: GSM4546574: wt1; Saccharomyces cerevisiae; OTHER
library_construction_protocol: Based on Pan et al. 2011 Cell, and Zhu and Keeney 2015 Genetics, with modifications. Whole cell extracts were prepared by grinding frozen cells in a bead mill and resuspending powder in 10%TCA. Pellets were then extracted with SDS and soluble protein was subjected to affinity purification of Spo11-Flag fusion with Protein-G Dynabeads. Protein was digested with proteinase K and Spo11-associated oligonucleotides were recovered by ethanol precipitation. Based on Pan et al. 2011 Cell, and Thacker et al. 2014 Nature. Oligonucleotides were tailed with ribo-G using rGTP and terminal transferase, then ligated to duplex DNA adaptors with a dC overhang using T4 RNA ligase 2. Complementary strands were synthesized with Klenow, then purified by denaturing polyacrylamide electrophoresis. Purified complementary strands were subjected to 3' tailing with ribo-G using rGTP and terminal transferase, then another duplex adaptor was ligated using T4 RNA ligase 2. Ligated material was amplified with 16 cycles of PCR to add Illumina HiSeq adaptors
identifiers.namespace: GEO
identifiers.id: GSM4546574