Cutana chic cut run kit

CUT&RUN was performed using CUTANA ChIC/CUT&Run Kit (Epicypher 14–1048) following manufacturer’s instruction in User Manual Version 2.1 or User Manual Version 3. Three technical replicates per antibody were performed. For unsorted, bulk samples, 5x10⁵ cells per replicate were harvested using versene (Thermo Fisher 15040066). For sorted state populations, cells were harvested using versene, and 8x10⁵ cells per replicate were isolated by flow cytometry. Harvested cells were permeabilized using 0.005% digitonin. Additionally, 1 ug of antibody per sample was added (Klf4 Abcam ab12947, Zfp281 Abcam ab10131), with H3K4me3 and Rabbit IgG antibodies (CUTANA 13–0041, 13–0042) used as the positive and negative controls, respectively. E.coli spike-in DNA was also added to each sample. For CUT&RUN qPCR, the resulting CUT&RUN DNA was quantified by Qubit and 0.11ng DNA was used per qPCR reaction. The percent input was calculated as 2^{ΔCT[input] – ΔCT[sample]}. For CUT&RUN followed by sequencing, library preparation using the NEB UltraII kit (NEB E7645) was performed on equal amount per replicate of the resulting CUT&RUN DNA.

CUT&RUN was performed sorted DN1, DN2, and DN3 cells using CUTANA^™ ChIC/CUT&RUN Kit (EpiCypher, Cat#14–1048), using manufacturer’s recommendation. Briefly, between 20,000–200,000 live cells were sorted and nuclei were extracted, washed, and allowed to adsorb onto activated Concanavalin A beads. Cells were then resuspended in recommended buffer, 0.5 mg of antibody was added, mixed well, and allowed to incubate at 4°C overnight on a nutator. Anti-TCF-1 (Cell Signaling Technology, C63D9) antibody was used along with positive and negative controls. Subsequently, the reactions were washed with cell permeabilization buffer and incubated with pAG-MNase, and DNA was isolated for the antibody-bound regions. At least two biological replicates were generated for each experiment. Library preparation was carried out using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) and were paired-end sequenced (38bp+38bp) on a NextSeq 550 (Illumina) or 61bp+61bp on Novaseq 6000 (Illumina).