GSM8994756: UTX_CUT&Tag_WT_CD8+ Progenitor_3; Mus musculus; OTHER
Sorted cells were performed Epicypher CUT&Tag protocol After sorting, nuclei were isolated with cold nuclear extraction buffer and incubated with activated concanavalin A-coated magnetic beads (Polysciences, 86057-3) in PCR strip tubes at room temperature for 10 min. A 1:100 dilution of primary antibody (anti-UTX Cell Signaling Rabbit monoclonal antibody no. 33510 or IgG isotype control: Cell Signaling Technology, 3900S) in antibody buffer was added and nuclei were incubated with primary antibodies overnight at 4°C. The next day, the strip tubes were incubated on a magnetic tube holder and supernatants were discarded. Secondary antibody (guinea pig anti-rabbit IgG; Fisher Scientific, NBP172763) was added diluted at 1:100 in Dig-Wash and nuclei were incubated for 1 h at room temperature. Nuclei were washed four times in Dig-Wash and then incubated with a 1:20 dilution of pAG–Tn5 adaptor complex (EpiCypher) in Dig-300 buffer for 1h at room temperature. To stop tagmentation, 25 μl Dig-300 buffer with 10 μl 1M MgCl, 7.5μl 0.5M EDTA, 2.5μl 10% SDS and 5μl 10mg ml−1 proteinase K was added to each reaction and incubated at 55°C for 1h. DNA was extracted by phenol:chloroform:isoamyl alcohol separation. DNA was barcoded and amplified using the following conditions: a PCR mix of 25μl NEBNext 2× mix, 2μl each of barcoded forward and reverse 10μM primers, and 21μl of extracted DNA was amplified at: 58°C for 5min, 72°C for 5min, 98°C for 4 s, 16× 98°C for 15s followed by 63°C for 10s, 72°C for 1min. Amplified DNA libraries were purified by adding a 1.3× volume of KAPA pure SPRI beads (Roche) to each sample and incubating for 10min at 23°C. Samples were placed on a magnet and unbound liquid was removed. Beads were rinsed twice with 80% ethanol, and DNA was eluted with 25 μl TE buffer. All individually i7-barcoded libraries were mixed at equimolar proportions for sequencing on an Illumina Novaseq X Plus sequencer with 50bp pair-end sequencing.