GSM2611277: Fbxl19flfl_UNT_Input_rep1; Mus musculus; ChIP-Seq
Chromatin immunoprecipitation was performed as described previously (Farcas et al., 2012) with slight modifications. Cells were fixed for 45 min with 2mM DSG (Thermo scientific) in PBS and 12.5 min with 1% formaldehyde (methanol-free, Thermo scientific). Reactions were quenched by the addition of glycine to a final concentration of 125µM. After cell lysis and chromatin extraction, chromatin was sonicated using a BioRuptor Pico sonicator (Diagenode), followed by centrifugation at 16,000×g for 20min at 4°C. 200µg chromatin diluted in ChIP dilution buffer (1% Triton-X100, 1mM EDTA, 20mM Tris-HCl (pH 8.0), 150mM NaCl) was used per IP. Chromatin was precleared with protein A Dynabeads blocked with 0.2 mg/ml BSA and 50 µg/ml yeast tRNA and incubated with the respective antibodies overnight at 4°C. Antibody-bound chromatin was purified using blocked protein A Dynabeads for 3 hours at 4°C. ChIP washes were performed as described previously (Farcas et al., 2012). ChIP DNA was eluted in ChIP elution buffer (1% SDS, 100mM NaHCO3) and reversed cross-linked overnight at 65°C with 200mM NaCl and RNase A (Sigma). The reverse cross-linked samples were treated with 20ug/ml Proteinase K and purified using ChIP DNA Clean&Concentrator™ kit (Zymo research). ChIP DNA material was sheared with BioRuptor Pico sonicator to obtain DNA fragments of average size 200-800 bp. ChIPseq libraries were prepared with  30-40ng of ChIP DNA material using NEBNext Ultra DNA Library Prep Kit.