GSM8765467: Thymic iNKT cells, Ilaros (ChIP); Mus musculus; ChIP-Seq
Thymocytes from iNKT-iPS cell-derived mice were resuspended in 2%FBS/RPMI1640 medium and stained with alpha-Galactosylceramide (a-GalCer)-loaded mouse CD1d tetramer-APC and anti-APC microbeads. Thymic iNKT cells were purified by MACS magnetic separator (Miltenyi). Total thymocytes from wild-type mice and purified thymic iNKT cells were washed with PBS and cell pellets were snap-frozen in liquid nitrogen. Frozen cells were sent to Active Motif for further analysis. Active Motif fixed cells and chromatin was extracted and applied to sonication after quenching. Active Motif performed ChIP reactions, library generation and barcoding for next-generation sequencing and data analysis. Spike-in of Drosophila chromatin was performed before ChIP for normalization. 30 ug of chromatin and 30 ul of anti-Ikaros antibody (Cell Signaling Technology, #14859) were used for the ChIP reaction and the reaction was validated by qPCR. Library generation, library quality check and barcoding were performed and next-generation sequencing was done using Illumina NextSeq 500. As an input, equal amounts of unprecipitated genomic DNA from each sample were used to generate a next-generation sequencing libraries, and sequenced in parallel with the ChIP-seq samples.