Active motif antibodies against Rpb1 (phosphoS5)(39097)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Crosslinking was performed with 1% formaldehyde for 10 min at RT and stopped by adding glycine. After washings, chromatin was lysed in SDS-containing buffer (50 mM HEPESKOH, pH 7.9; 140 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na deoxycholate, 0.1% SDS) with protease inhibitors cocktail (Roche) and sheared to 500-bp fragments by sonication. Sonicated chromatin was centrifuged twice at 16 000 g for 20 min, and used in immunoprecipitation experiments. For one experiment, about 10 ?g of antibodies and 8 ?L of MabSelect sepharose (GE healthcare) were taken; BSA were added to a final concentration of 1%. The precipitated chromatin was sequentially washed twice with SDS-containing buffer and with TE buffer (20mM Tris-HCl, pH 8.0; 1mM EDTA). Precipitated chromatin complexes were eluted by sequential incubation in two portions of elution buffer (50mM Tris-HCl, pH 8.0; 1mM EDTA, 1% SDS), 30 min each, at room temperature. The eluted chromatin solution was supplemented with 5 M NaCl (16 ?L per 500 ?L sample) and incubated at 65°C for 16 h on a thermoshaker for de-crosslinking. De-crosslinked chromatin was subjected to proteinase treatment (3 ?L of proteinase K and 5 ?L of 0.5M EDTA per 500 ?L sample) and incubated at 55°C for 4 h on thermoshaker. DNA was extracted with a phenol/chloroform mixture and precipitated with isopropanol. The precipitate was dissolved in TE buffer and subjected to library preparation. Mnase-ChIP seq was performed with the same protocol except one stage. Step of chromatin sonication was change to treatment with Micrococcal nuclease (ThermoScientific) for 5 min at 37°C and for 30 min at 4°C. DNA libraries were prepared for sequencing using standard Illumina protocols