title: GSM3399491: H3K27me3_RA; Mus musculus; ChIP-Seq
library_construction_protocol: 6x105 low passage (< 10) E14 cells were plated per 15 cm culture dish and cultivated for 48 h in regular ES medium containing LIF. 4 h prior to cross-link cells were treated with LIF or RA as described in the treatment protocol. Then cells were washed with PBS, trypsinized and gently but thoroughly resuspended in ES medium to obtain single cell suspension. Cells were cross-linked adding 1% formaldehyde for 5 min and the reaction was quenched using 125 mM Glycin for another 5 min. After two wash steps with PBS, cell pellets were resuspended in 100 µl sonication buffer (20 mM Tris-HCl pH 7.5, 2 mM EDTA pH 8.0, 1% Triton X-100, 150 mM NaCl, 0.1% SDS and Complete proteinase inhibitor cocktail (Roche)) per 106 cells and chromatin was sheared by sonication applying a Bioruptor Pico (Diagenode) device for 25-35 cycles. Automatic ChIP was performed using the SX-8G Compact IP-Star liquid handler (Diagenode) in combination with Auto Histone Kits (Diagenode, C01010022). Using the pre-programmed method 'indirect ChIP', ChIP reactions were carried out in a final volume of 200 μl for 10 h followed by 5 h beads incubation and 5 min washes (at 4°C). After ChIP, eluates were recovered, RNase A-treated, decrosslinked ON at 65°C and treated with Proteinase K for 4 h at 55°C. The recovered DNA was purified using ChIP DNA Clean & Concentrator Kit (Zymo research, D5205). Sequencing libraries were prepared using the NEBNext® UltraTM DNA Library Prep Kit (NEB, E7370) according to manufacturer's instructions.