title: GSM5848406: CUTRUN_NFIA_HUDEP2 clone 6_NFI-451; Homo sapiens; OTHER
library_construction_protocol: 0.5-1 million undifferentiated HUDEP2, HUDEP2 undergoing 2 days of differentiation, and primary cells undergoing 8 days of erythroid differentiation were used for CUT&RUN. We followed the experimental procedure published by Henikoff lab (dx.doi.org/10.17504/protocols.io.bcuhiwt6) with adaptations15. Antibody incubation was performed in 300μl PCR tubes for 2 hours in the cold room. pA/G-MNase and Spike-In DNA (#40366) were obtained from Cell Signaling. The following antibodies were used: IgG (#3900, Cell signaling), BCL11A (ab191401, Abcam), NFIA (PA5-52252, Thermo Fisher Scientific), NFIX (SAB1401263, Sigma Aldrich), GATA1(Ab11852, Abcam), H3K27Ac (MA5-23516, Invitrogen). All antibodies were used approximately 1μg in 100μl. The concentration of the released DNA fragments was determined by Qubit and 15-30ng DNA was used for adapter ligation and library construction. The sequencing libraries were generated using the kit from NEB (Cat# E7645 and E6440S). We followed the experimental procedure posted by Nan Liu (dx.doi.org/10.17504/protocols.io.wvgfe3w) to size select the <120 bp fragment and facilitate the transcription faction binding site identification20. The multiplexed sequencing libraries were pair-end (2x50bp) sequenced on Illumina Nextseq 2000 platform.