title: GSM6153701: Day7 Malat1 Knockdown no input, ChIP-seq; Mus musculus; ChIP-Seq
library_construction_protocol: Spleens from P14 mice were homogenized, and 1x106 cells/mL in TCM were pulsed with 1 ng/ml LCMV GP33–41 peptide for 1 hour at 37°C. Cells were washed once with equal volume of warm TCM, then plated in a 96 well-plate at 5x104 cells/well. Cells were harvested 4.5 days later and dead cells removed using the Dead Cell Removal kit according to the manufacturer's protocol (Miltenyi Biotec, Catalog number: 130-010-101). CD8+ T cells were then enriched using the CD8a+ T Cell Isolation Kit. Chromatin was then prepared using the EMD Millipore Magna ChIP A/G Chromatin Immunoprecipitation Kit according to manufactures protocol (Catalog Number: 1710085) and flash frozen in liquid nitrogen. Nuclei were sheared in Covaris microTUBES (Catalog number: 520045) using the Covaris E220 (Peak Incident Power 175W, Duty Factor 10%, Cycles per Burst 200, Treatment Time 600 seconds). For each immunoprecipitation (IP), 3 mg antibody per 1e6 cell equivalents were used for overnight incubation at 4°C. Antibodies used for IPs were as follows: anti-H3K27me3 (07-449, EMD Millipore), anti-H3K4me3 (ab8580, abcam). and anti-Ezh2 (AC22, 07-449, EMD Millipore). Five percent of each sample was kept as input control.