Illumina HiSeq 2000 sequencing; ChIP-seq analysis identifies p27(Kip1)-target genes involved in cell adhesion and cell signalling in mouse embryonic fibroblasts
ChIP-seq analysis identifies p27(Kip1)-target genes involved in cell adhesion and cell signalling in mouse embryonic fibroblasts
Sample 1_s
For these experiments mouse embryonic fibroblasts (MEFs) from p27WT and p27-/- animals were used. Cells were cultured in Dulbecco's modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Biological Industries), 2 mM L-Glutamine (Merck), 1% nonessential amino acids (Biological Industries), 1 mM pyruvic acid (Sigma), 50 U/ml penicillin and 50 μg/ml streptomycin (Biological Industries). Prior to synchronization cells were cultured up to 90% confluence and to make them quiescent they were maintained for 72h in the medium in the absence of serum. Cell synchronization was monitored by flow cytometry of propidium iodide-stained cells. DNA was extracted from samples by ChIP-seq. Quiescent MEFs were used for ChIP with anti-p27 antibodies (Santa Cruz SC528). Briefly, cells were lysed and chromatin from cross-linked cells was sonicated. Chromatin was incubated with 5 µg of anti-p27 in RIPA buffer (50 mM Tris−HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 0.1 mM Na3VO4, 0.5 μg/μl aprotinin, 10 μg/μl leupeptin) adding 20 µl of Magna ChIP Protein A magnetic beads (Millipore). Samples were incubated in rotation overnight at 4ºC. Beads were washed with low salt buffer, high salt buffer, LiCl buffer and TE buffer. Subsequent elution and purification of the immunoprecipitated DNA-proteins complexes was performed using the IPure kit (Diagenode) according to manufacturer’s protocol. After DNA isolation, ChIP samples, together with the input, were sequenced. Libraries were prepared using the NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina® kit (ref. E6200S) according to the manufacturer's protocol. Briefly, 10 ng of input and ChIP enriched DNA were subjected to end repair, addition of “A” bases to 3′ ends and ligation of PE adapters. All purification steps were performed using Qiagen PCR purification columns (refs. 50928106 and 50928006). Library size selection was done with 2% low-range agarose gels. Library amplification was performed by PCR on the size selected fragments. Final libraries were analyzed using Agilent DNA 1000 chip to estimate the quantity and check size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit (ref. KK4835, KapaBiosystems) prior to amplification with Illumina’s cBot. Sequencing was done on the HiSeq2000, Single Read, 50 nts. Libraries were prepared using the NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina® kit (ref. E6200S) according to the manufacturer's protocol. Briefly, 10 ng of input and ChIP enriched DNA were subjected to end repair, addition of “A” bases to 3′ ends and ligation of PE adapters. All purification steps were performed using Qiagen PCR purification columns (refs. 50928106 and 50928006). Library size selection was done with 2% low-range agarose gels. Library amplification was performed by PCR on the size selected fragments.