title: GSM3004381: (6-4)PP_XR-seq_5M_rep2; Saccharomyces cerevisiae; OTHER
library_construction_protocol: To isolate the excised oligomers carrying UV photoproducts in vivo, we first tested 3 different strategies to disrupt yeast cell walls and lyse cell membranes: 1) Cells were pelleted and resuspended in 300 µL STES (500 mM NaCl, 200mM Tris-Cl, 10 mM EDTA and 0.1% SDS), 300 µL phenol:chloroform:isoamyl alcohol (25:24:1) and 600 µL acid washed glass beads. 2) The same as the aforementioned method except STET (500 mM NaCl, 200mM Tris-Cl, 10 mM EDTA and 1% Triton X-100) was used instead of STES. 3) Cells were pelleted and resuspended in 600 µL TEP (150 mM Tris-Cl, 30 mM EDTA and 10 µL Proteinase K) and 600 µL acid washed glass beads. All the resuspension were bead beat on a Mini-Beadbeater-16 (Bio Spec) for 2.5 min at 4°C. For samples containing STES or STET buffer, the cell lysates were then centrifuged, and the supernatants were sequentially treated with 5 µL RNase A at 37°C for 1 h and 5 µL Proteinase K at 55°C for 1 h. Samples were then extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with ethanol. The precipitated small DNA molecules were further purified by a G-50 filtration column (GE Healthcare) and subjected to immunoprecipitation with anti-(6-4)PP antibody as described previously. For samples containing TEP buffer, 66 µL 10% SDS was mixed with the disrupted cells, and 165 µL 5M NaCl was mixed in after incubation at room temperature for 10 min. Following overnight incubation at 4°C, the supernatant fraction was separated by centrifugation, incubated with 10 µl RNase A at 37°C for 1 h and then 5 µl proteinase K at 55°C for 30 min. The excised oligomers were then sequentially purified by phenol-chloroform extraction, ethanol precipitation, G-50 column filtration and DNA damage immunoprecipitation. This latter approach approximates the Hirt lysis method used previously. For direct excision assay, the excised oligomers isolated by any of the three different methods were 3' end labeled with 32P-Cordycepin and resolved in 11% denaturing sequencing gel. The results indicate that the strategy of cell wall disruption followed by Hirt extraction efficiently isolates the excised oligomers. Excised oligomers, purified by the G-50 column filtration followed by anti-CPD or anti-(6-4)PP antibody immunoprecipitation, were ligated to adaptors as described previously. The ligated products were then subjected to a second round of DNA damage-specific immunoprecipitation. The CPDs and (6-4)PPs were then repaired by photoreactivation using CPD photolyase or (6-4)PP photolyase as described. One percent of the unrepaired and repaired ligation products were used in sets of PCR amplification reactions to determine the minimum number of PCR cycles needed for the final XR-seq library preparation. The number of PCR cycles used for CPD XR-seq and (6-4)PP XR-seq in this study ranged from 12 to 14. Libraries were gel-purified, pooled and sequenced using the Illumina Hiseq 2500 platform.
attributes.tag: GEO Accession
attributes.value: GSM3004381