GSM2536329: IgG WT ChIP-Seq; Mus musculus; ChIP-Seq
At 2.5 dpc (days post coitus), females were sacrificed and checked for presence of preimplantation embryos by flushing the oviduct. Whole uterus were immediately cleaned of fat and cut into halves and pieces from each female were cross linked with 1% paraformaldehyde in PBS at room temperature for 10 minutes. Then, they were placed in 0.125 M glycine for 5 minutes to stop cross-linking. Subsequently, samples were washed in cold PBS twice and then homogenized to single cells using mechanical shearing of tissue in lysis buffer without detergent (SDS). Cells were pellleted at 1200 rpm for 5 minutes and then supernatant aspirated. Pellet was resuspended in lysis buffer with detergent (SDS) to release chromatin for 10 minutes at room temperature. Loose chromatin was pelleted at 1500 rpm for 5 minutes and supernatant discarded. Pellet was resuspended in ice-cold sonication buffer and subjected to sonication using Diagenode Picoruptor. Sonication efficiency is checked on 1% agarose gel upon decrosslinking and sheared chromatin is obtained by spinning at 20000G/4C/15 mins to pellet down non-chromatin fragments. Pure chromatin is measured for protein concentration and stored at -80 C till immunoprecipitation. Protocol followed manufacturers instructions for ChiPSeq libraries using the NEBNext Ultra II DNA library prep kit (Illumina, #E7645)