Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J (Cas9) x C57BL/6-Tg(Bcl2)25Wehi/J (Bcl2Tg)
treatment
Transduced with sgPU.1-CFP
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
Cell isolation: Fetal livers (FL) were disected from E13.5 (day of plug, E0.5) C57BL/6 animals. Suspensions of FL cells were then prepared, stained for lineage markers using biotin-conjugated lineage antibodies (CD11c, Gr1, TER-119, NK1.1, CD19, F4/80), incubated with streptavidin-coated magnetic beads (Miltenyi Biotec), and passed through a magnetic column (Miltenyi Biotec). Lineage-depleted (Lin−) cells were eluted and stored in liquid nitrogen in freezing media (50% FBS, 40% αMEM, 10% DMSO) for future use. Cell culture: Frozen lineage depleted FL cells from E13.5 were thawed and used to initiate OP9-DL1 cultures in OP9 medium (α-MEM, 20% FBS, 50 μM β-ME, Pen-Step-Glutamine) supplemented with IL-7 and Flt3L (5 ng/ml each). Cells were incubated at 7% CO2 and 37°C. Scid.adh.2C2 cells were cultured in RPMI1640 with 10% fetal bovine serum (Sigma-Aldrich), sodium pyruvate, non-essential amino acids, Pen-Strep-Glutamine and 50 μM β-mercaptoethanol. Cells were incubated at 5% CO2 and 37°C. FACS staining. For sorting of transduced CD25 enriched FLP derived cells, surface antibodies against c-Kit, CD25, CD44, CD45, Mac1/CD11b were used for staining of Lin- FLP derived CD25+ cells (7AAD-CD45+GFP+CD25+) (EVGFP, PU1WTHA, PU1ENGHA and PU1ETSHA) or CD44+CD25 cells (7AAD-CD45+GFP+CD44+CD25-) (PU1WTHA). Transduced Scid.adh.2c2 cells for Lzr-PU.1 ChIP and RNA-sequencing were sorted as LiveGFP+. For sorting for primary cell ATAC experiment, thymocytes were pre-depleted with biotin-conjuagated a-CD8a, a-TCRrd, a-TCRb, a-Ter119 followed by MACS magnetic column purifcation as described above. Depleted thymocytes were stained with CD45, CD44, c-Kit, CD25, and a biotin-conjugated lineage cocktail (CD8a,CD11b,CD11c,CD19,Ly-6G(Gr-1),NK1.1,TER119,TCRrd,TCRb) and were sorted for ETP progenitors (Lin−CD45+c-KithiCD44hiCD25−) or DN3 progenitors (Lin−CD45+c-KitlowCD44lowCD25+).