Mnase

For HDAC3 qChIP, DP thymocytes were enriched using the EasySep Mouse Streptavidin RapidSpheres Isolation kit (#19860, StemCell Technologies) to remove SP and DN thymocytes with biotin-conjugated anti-CCR7 (4B12), anti-IL-7Rα (A7R34), anti-H2K (AF6-88.5), anti-CD44 (IM7), and anti-CD25 (PC61.5) as well as anti-TCRγδ (UC7-13D5) antibodies. For SP thymocyte enrichment, Mouse PE Positive Selection kit (#18554, StemCell Technologies) was used positively select for CCR7⁺ thymocytes (PE-conjugated anti-CCR7 (4B12)). ChIP was performed according to Pchelintsev et al. (2016) (link), with the following adjustments: After cell lysis and brief sonication (four mins, 30 s on/30 s off; Bioruptor Pico (Diagenode, Inc.)), equal volume of 2x MNase buffer (35 mM Tris-HCl pH 7.5, 25 mM NaCl, 120 mM KCl, 2 mM CaCl₂) was added to each sonication sample along with 3.75 U/μl of MNase (Cell Signaling Technologies #10011S) at 37°C for 15 mins. Anti-HDAC3 antibody (#85057, Cell Signaling Technologies) was used to isolate chromatin bound to HDAC3 after chromatin fragment size was confirmed to be less than 500 bp by gel electrophoresis. Isolated DNA was used to perform real-time PCR. Graphs depict fold enrichment over regions without HDAC3 binding (Rpl30 primers, Cell Signaling Technologies (#7015)). See Supplementary file 1 for primer sequences.

ChIP assay was performed as described by Liu et al. [5 (link)]. Briefly, cells were lysed and treated with MNase (Cell Signaling, Beverly, MA, USA) to obtain DNA fragments of 300–800 bp that were then immunoprecipitated with target antibodies (anti-trimethyl-Histone H3 Lys27 and rabbit IgG) at 4 °C overnight. Extraction of immunoprecipitated DNA fragments using protein-A sepharose (Sigma-Aldrich) followed next prior to purification using DNA purifying slurry (Diagenode, Denville, NJ, USA). About 200 ng of purified DNA template was then amplified real-time PCR. Promoter-specific primers were used and are listed below (Table 3). Data was expressed as a percentage of the input DNA.