GSM9687350: Tfr, WT, rep1, CUT&Run; Mus musculus; OTHER
CUT&RUNs were performed following the EpiCypher protocol with minor modification. From ~300,000 sorted T cells, nuclei were isolated using nuclear extraction buffer (20 mM HEPES-KOH [pH 7.9], 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 0.5 mM spermidine, and protease inhibitors (GenDEPOT, Altair, Texas, USA; P3100-010)) in ice for 10 minutes. The pelleted nuclei were resuspended in CUT&RUN wash buffer (20 mM HEPES-KOH [pH 7.5], 150 mM NaCl, 0.01 % Digitonin (Merck Millipore, Burlington, Massachusetts, USA; 300410-250MGCN), 0.5 mM spermidine, and protease inhibitors). Nuclei were bound to CUTANA concanavalin A-conjugated paramagnetic beads (EpiCypher, Durham, North Carolina, USA; 21-1401) that had been washed with bead activation buffer (20 mM HEPES-KOH [pH 7.9], 10 mM KCl, 1 mM CaCl2, and 1 mM MnCl2) at room temperature for 10 minutes. The ConA-bead-bound nuclei were then incubated at 4 ℃ on a nutator overnight in CUT&RUN antibody buffer (CUT&RUN wash buffer supplemented with 2 mM EDTA [pH 8.0]) and antibodies anti-CEBPa (Cell Signaling Technologies, Danvers, Massachusetts, USA; #8178), IgG antibody (EpiCypher, 13-0042). The Con-A-bead-bound nuclei were washed and resuspended in CUT&RUN wash buffer. The Con-A-bead bound nuclei were treated with 20X pAG-MNase (EpiCypher, 15-1116) and incubated at 4 ℃ on a nutator for 1 hour. The Con-A-bead bound nuclei were washed and resuspended in CUT&RUN Triton-wash buffer, followed by the addition of 100 mM CaCl2 (final concentration 2 mM), and incubated at 4 ℃ on a nutator for 2 hours. 2X STOP buffer (340 mM NaCl, 20 mM EDTA [pH 8.0], 4 mM EGTA [pH 8.0], and 0.2 μg/μl RNase A (Thermo Fisher Scientific, Waltham, Massachusetts, USA; EN0531)) was added to stop chromatin digestion and incubated at 37 ℃ for 20 minutes. The Con-A-bead bound nuclei were captured using a magnet, and supernatants were collected. The fragmented DNA was treated 1.6 U proteinase K (NEB, P8107S) containing 0.1 % SDS at 65 ℃ for 1 hour. The DNA was purified using phenol:chloroform:isoamyl alcohol (Thermo Fisher Scientific, 15593) followed by ethanol precipitation. The DNA was resuspended in 1X TE (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA [pH 8.0]) buffer. CUT&RUN library construction was performed using NEBNext Ultra™ II DNA Library Prep Kit for Illumina (NEB, E7645L) following the manufacturer's instructions with minor modifications. Briefly, the end-prepped DNAs were ligated with adaptors. The adaptor-ligated DNAs were digested with USER enzyme and size selected using 0.6X and 1.4X SPRIselect beads (Beckman Coulter, Brea, California, USA; B23317). Size-selected adaptor-ligated DNAs were amplified by 14 ~ 15 cycles of PCR. The amplified CUT&RUN library was size-selected using 1.4X SPRIselect beads and proceeded to sequencing. CUT&RUN library samples were sequenced on Illumina NovaSeq-X (Illumina) with 100bp paired-end reads.