GSM5600936: Mutant at reprogramming day 2; Mus musculus; ChIP-Seq
All ChIP experiments were performed with ChIP-IT Express Chromatin Immunoprecipitation kit (Active Motif), following the kit protocol with minor modifications. Briefly, day 10 reprogramming cells (6.0X105 seeding density) in a 60-mm dish were crosslinked with 0.09 mL of 37% formaldehyde to 3.3 mL of growth medium. 1.5 mL of glycine (20X) was added to quench the unreacted formaldehyde. Cells in each 60-mm plate were collected and resuspended in 0.2 ml of lysis buffer. Genomic DNA was sheared to a targeted length of 150-200 bp on Covaris S220 Focused-ultrasonicator with optimized conditions. Sheared chromatin was subjected to cross-link reversal, treated with Proteinase K and RNase A, then phenol/chloroform extracted and analysed for the shearing efficiency on 1% agarose gel electrophoresis. For KLF4 ChIP ~3.2X106 reprogramming cells and 5 mg of antibody were used for each experiment. Finally, DNA fragments were purified using Chromatin IP DNA Purification kit (Active Motif) following the manufacturer's protocol and used for real time qPCR and ChIP-seq library preparation. The primary antibodies used as follows: Anti-Human KLF4 (R&D, AF3640). ChIP-seq library was generated using KAPA Hyper Plus kit (Roche) by Lab Droid “Maholo” (Robotic Biology Institute) automatically.