GSM5061428: TS_IP; Mus musculus; ChIP-Seq
US/VOT-E36 cells (University of Sheffield/ventral otocyst-epithelial cell line clone 36) were used, derived from the otocyst of Immortomice at embryonic day 10.5 (Lawoko-Kerali et al., 2004). The cells were cultured in minimum essential medium (Gibco) supplemented with 10% fetal bovine serum and 50 U/ml -IFN in a humidified atmosphere with 10% CO2 and at a temperature of 33oC, conditions under which these cells proliferate in the absence of differentiation. For the ChIP assay, once the cultures had reached 85-90% confluence, the chromatin from the cells in six 75-cm2 flasks was cross-linked in 1% formaldehyde for 10 minutes at room temperature; next, the reaction was quenched by adding glycine to a final concentration of 125mM and mixing during an additional 5 minutes. Following removal of the medium and various washes with phosphate buffer saline, cells were scraped off the tissue culture flasks, pelleted and lysed; the chromatin was then sheared into 200-500 bp fragments by sonication (using pulses of 30 seconds on and 30 seconds off in a water-bath sonicator). Any remaining cellular debris was subsequently removed by centrifugation and the sonicated chromatin was pre-cleared for 4 hours at 4oC with protein A agarose beads (Roche). After this time, the beads were removed by centrifugation; half the volume of the chromatin sample was immunoprecipitated (IP sample) at 4oC overnight with a 1:1 mixture of an antibody against Meis2 (K846) and K830, an antibody that recognizes both Meis 1a and Meis 2a isoforms (Penkov et al., 2013). A volume that was equivalent to one tenth of the volume used for the IP reaction was also at that time set apart and frozen, constituting the Input control sample. Following immunoprecipitation with the Meis1/2 antibodies, beads that had been pre-blocked at 4oC overnight with BSA and a rabbit IgG isotype control (ChromPure Rabbit IgG, Jackson ImmunoResearch Laboratories) were added to each tube; the samples were then incubated at 4oC for 4 hours. Afterwards, a series of washes using multiple buffers were conducted in order to remove any molecule that had bound non-specifically to the beads; the chromatin was finally eluted from the beads and cross-linking to the antibody reversed by incubating the IP sample in the presence of NaCl at 65oC overnight; the Input sample that had been stored at -20oC was also incubated in the same solution at 65oC. The next day, both the IP and the Input chromatin were purified using a PCR purification kit (Qiagen), following manufacturer´s instructions. The samples were then used to carry out ChIP-seq analysis. 0.5 ng of total DNA for both Input and IP were used to generate barcoded libraries using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (New England Biolabs). Basically, adapters are ligrated to DNA followed by an amplification and clean up. The size of the libraries was checked using the Agilent 2100 Bioanalyzer High Sensitivity DNA chip and their concentration was determined using the Qubit® fluorometer (ThermoFisher Scientific). Libraries were applied to an Illumina flow cell for cluster generation (SR Cluster Kit cBot) and sequence-by-synthesis single reads of 60 base length using the SBS Kit (Illumina) were generated on the HiSeq 2500 following the standard sequencing protocol. Reads were further processed using the bcl2fastq package (Illumina) to split reads according to adapter indexes and produce fastq files. RNA-Seq was performed at the Genomics Unit of the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid.