GSM3109113: EFT; Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344; ChIP-Seq
To immunoprecipitate StdE-DNA and StdF-DNA complexes, 800 μL of chromatin, 20 μL of Ultralink Immobilized protein A/G beads (Pierce) and 2 μL of the corresponding antibody were used (anti-StdE and anti-FLAG). Control sample (mock-IP) was performed with no antibody added. They were then incubated for 90 minutes at room temperature on a rotating wheel. Beads were transferred to a Spin-X column tube (Costar) and centrifuged at 3000rpm for 1min. Beads were gently re-suspended in 500 μL of IP buffer and incubated on wheel for additional 3min. Beads were washed with 500 μL of IP salt buffer, IP wash buffer and TE pH8.0 by resuspending and centrifuging the sample. The column was transferred to a fresh tube and the beads were resuspended in 100 μL of elution buffer (50 mM Tris-HCl at pH 7.5, 10 mM EDTA, and 1% SDS) and incubated at 65ºC for 20min. After centrifuging at 3000rpm for 1min, the flow-through was treated with 10μL of 40mg/mL Pronase (Roche) made up in TBS. The samples were heated at 42ºC for 2h and 65ºC for 6 hours. The reactions were then kept at 4ºC overnight. The samples were cleaned up by using a PCR clean up Kit (Promega) and resuspended in 50 μL of H2O. NEBNext® Ultra DNA Library Prep Kit for Illumina; 50 nt SE sequencing.
GEO Accession
GSM3109113