title: GSM8093273: ChIPseq Ikaros 0h Rep2; Mus musculus; ChIP-Seq
library_name: GSM8093273
library_construction_protocol: Cells were fixed at room temperature in 10ml PBS with 1mM DSG (ThermoFisher 20593) for 30 min followed by addition of 1% formaldehyde (ThermoFisher 28908) and incubated for a further 10min. The reaction was quenched with 125mM glycine. Chromatin was digested by sonication or MNase to obtain fragments between 100-300bp. A small aliquot was reserved for the Input sample, and chromatin equivalent to 4 million cells was used per IP. HA-Ikaros-ERt2 ChIP performed using overnight incubation with Pierce magnetic anti-HA beads (Pierce 88836). For H3K27ac (Active Motif 39133) and CHD4 (Abcam ab70469), 4-6 mg of antibodies were incubated with each reaction overnight, followed by 2h of incubating with magnetic Prot A/G Dynabeads (ThermoFisher 10001D, 10003D). Washes were performed for 5 min rotating in the cold room twice with each buffer, Low Salt Buffer, High Salt Buffer, LiCl Buffer and TE. DNA was eluted with 1% SDS and 0.1M NaHCO3. For reverse crosslink, NaCl was added to a final concentration of 0.2M, and samples were incubated at 65°C shaking overnight, following by 2h of RNaseA and 2h of Proteinase K treatment. NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (E7645S) was used for all ChIP-seq libraries with PCR amplification of 5-8 cycles. Clean-up was performed with Ampure beads and quantification and quality control of libraries was performed using DNA Qubit and HS DNA Bioanalyser.