GSM7432142: GATA2 (pooled samples); Homo sapiens; OTHER
Cut&Run experiments will be performed according to the published protocol (1, 2). hTSC were cultured in the stem condition following published protocol (3). When cells reached about 70% confluency, cells were washed with room temperature PBS and trypsinized to get single cell suspension. 500,000 hTSC cells were used per condition. Briefly, 500,000 hTSC cells were washed, then mixed with Concanavalin A beads. The immobilized cells were incubated with the primary antibody GATA2 (Rabbit Abcam ab109241 and GATA3 (Mouse BD Pharmingen 558686) or IgG (Rabbit ab46540, Abcam) in a binding buffer containing digitonin. Immobilized cells on concanavalin A beads were washed with DIG wash buffer and then incubated with protein A/G-MNase (homemade). After washing these cells, MNase was activated by treatment with Ca2+ at 4°C for 30 minutes. Reactions were stopped by adding stop buffer containing RNase A and Glycogen blue. DNA fragments diffuse out of the cell, reaction mix were incubated at 37°C for 10 minutes without agitation. The soluble DNA fragments were transferred into a fresh tube to incubate with proteinase K at 55°C for 30 minutes. Finally, DNA fragments were purified and used directly for library preparation using the Illumina TruSeq ChIP. 1. Skene PJ, Henikoff JG, Henikoff S. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc. 2018 May;13(5):1006-1019. doi: 10.1038/nprot.2018.015. Epub 2018 Apr 12. PMID: 29651053. 2. Meers MP, Bryson TD, Henikoff JG, Henikoff S. Improved CUT&RUN chromatin profiling tools. Elife. 2019 Jun 24;8:e46314. doi: 10.7554/eLife.46314. PMID: 31232687; PMCID: PMC6598765. 3. Okae H, Toh H, Sato T, Hiura H, Takahashi S, Shirane K, Kabayama Y, Suyama M, Sasaki H, Arima T. Derivation of Human Trophoblast Stem Cells. Cell Stem Cell. 2018 Jan 4;22(1):50-63.e6. doi: 10.1016/j.stem.2017.11.004. Epub 2017 Dec 14. PMID: 29249463. For library preparation TruSeq RNA Library Prep Kit v2 was used. Finally, deep sequencing was performed using the Illumina NovaSeq 6000 sequencing system at the University of Kansas Genome Sequencing Facility to generate pair-end reads with read length 101 bp.