title: GSM5979950: WTKlf3MEFs Klf3 ChIP-seq Rep 1 INPUT; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP experiments were performed as previously described (Schmidt et al. 2009). Approximately, 7x107-1x108 cells were used for each ChIP. Cells were crosslinked with 1% formaldehyde for 10 min and the reaction was quenched by 2.5 mM glycine. Fixed cells were washed with cold PBS and centrifuged at 2000 x g for 5 min. Fixed cells were sonicated at high voltage for 20 min (30 seconds on, 30 seconds off) using a Bioruptor (Diagenode) to obtain 200-300 bp DNA fragments. The fragmented chromatin was pulled down at 4°C overnight using an antibody against V5 (Life Technologies, #R960CUS) or WDR5 (Australian Biosearch, #13105S). Chromatin cross-linking was then reversed at 65°C overnight followed by DNA purification. Real-time qPCR was performed on ChIP DNA on an Applied Biosystems ViiA7 Real-time PCR system (Applied Biosystems) to confirm the success of the ChIP experiments before sending to next generation sequencing. TruSeq DNA Sample Prep Kit