GATA1_HiChIP of human: Erythroid Day5, biological rep2
For GATA1 HiChIP, ten million Day-5 erythroid progenitors / Day-12 erythroblasts generated from ex vivo CD34+ erythroid differentiation culture were used. In brief, after fixation with 1% formaldehyde, cells were digested for 2 hours at 37C using 375U of MboI (NEB, Cat. #R0147). After biotin filling, proximity ligation was carried out with 4000U T4 DNA Ligase (NEB, Cat. # M0202M) for 2 hours at 25C. Sonication of re-suspended nuclei was performed by Covaris M220 sonicator. Following an overnight incubation with anti-GATA1 antibody (abcam, Cat.# ab11852), 100 L Protein A Magnetic Beads were added at 4C and incubated for 2 hours. Bound DNAprotein complexes were eluted and reverse-crosslinked after a series of washes.After reverse-crosslinking, ligation fragments containing biotin were immobilized on MyOne Streptavidin C1 beads (ThermoFisher Cat.#: 65602), and the library was prepared using the Tn5 transposome DNA library preparation kit (Vazyme, Cat.# TD502) as per the manufacturers protocol. DNA was then subjected to double-size selection using DNA Clean Beads (Vazyme, Cat. # N411) in order to isolate fragments between 300 and 600bp. Two biological replicates per condition were sequenced in a NovaSeq 6000 (S4, PE150, Illumina) by Novogene (Beijing).