title: GSM6457552: EB_CD71_CTCF_KO_CTCF_2; Mus musculus; ChIP-Seq
library_name: GSM6457552
library_construction_protocol: To produce CD71+ erythroid cells in vitro from differentiated mESCs (WT or mutant): 24h prior to differentiation, mESCs were induced by passaging into IMDM base media, trypsinised and plated in differentiation media (IMDM suplemented serum and other reagents and growth factors) in either triple vent petri dishes (Thermo Fisher) or flat-bottom 96-well plates (Thermo Fisher) at 3x104 cells in 10 cm dishes or 250 cells per well of a 96-well plate for up to seven days without disruption. EBs were harvested and disaggregated in 0.25% trypsin for 3 minutes at 37°C. CD71-positive cells were isolated by magnetic column separation (LS Column, Miltenyi), according to the manufacturer's instructions. Briefly, cells (from disaggregated EBs) were labelled with anti-mouse CD71-FITC (eBioscience 11-0711-85; 1:200) in staining buffer (PBS with 10% FCS; 500 ul per 10^7 cells) for 20 minutes at 4°C, washed, then incubated with MACS anti-FITC separation microbeads (Miltenyi; 10 ul  per 10^7 cells, following the manufacturer's instructions) and bead-labelled cells were retained by LS columns. Cells were then processed for ChIP-Seq as in Hay D et al, Nature Genetics, 2016. Cells were fixed with 1% Formaldehyde, lysed, sonicated and immunoprecipitated with the antibody of interest (CTCF: Merck Millipore 07-729 at 5ug/ml, H3K27me3: Cell Signaling 9733 at 2ug/ml, H3K4me3: Abcam 8580 at 2.5ug/ml). Libraries were prepared using the NEB Ultra II library preparation kit according to manufacturer's instructions. Samples were sequenced using 40 bp paired end reads on the NextSeq platform. The strategy was to characterise the chromatin state of primary mouse-derived or the CD71+ erythroid cells (WT and mutant) derived from mESC-EB in vitro differentitation system by isolating and analysing DNA regions bound by the antibodies of interest, genome-wide.