Epigenome mapping by ChIPmentation for H3K4me2

ChIPmentation was performed as previously described^{16, 39}, with minor adaptions. In each experiment, a maximum of 50,000 sort-purified cells were washed once with PBS and fixed with 1% paraformaldehyde for 10 min at room temperature. Glycine (0.125 M final concentration) was added to stop the reaction. Cells were collected at 500 g for 10 min at 4 °C (subsequent work was performed on ice and used cooled buffers and solutions unless otherwise specified) and washed once with ice-cold PBS supplemented with 1 mM phenylmethyl sulfonyl fluoride (PMSF). After centrifugation, cells were lysed in sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.25% SDS, 1x protease inhibitors (Sigma-Aldrich), and 1 mM PMSF) and sonicated (Covaris S220) for 30 min in a microTUBE until the size of most fragments was in the range of 200 to 700 bp. Following sonication, the lysate was adjusted to RIPA buffer conditions (final concentration: 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1x protease inhibitors (Sigma-Aldrich), and 1 mM PMSF).

For each immunoprecipitation, 10 μl magnetic Protein A (Life Technologies) were washed twice and resuspended in PBS supplemented with 0.1% BSA. 1 μg of antibody recognizing H3K4me2 (Aigma-Aldric, clone AW30) was added and bound to the beads by rotating overnight at 4 °C. Beads were added to the sonicated lysate and incubated for 2 h at 4 °C on a rotator followed by washing the beads once with RIPA low-salt buffer (10 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, and 1 mM EDTA), once with RIPA high-salt buffer (10 mM Tris-HCl, 500 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, and 1 mM EDTA), once with RIPA lithium-chloride buffer (10 mM Tris-HCl, 250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, and 1 mM EDTA), and once with Tris-Cl pH 8. Bead-bound chromatin was then resuspended in tagmentation mix (5 μl 5xTD buffer, 1 μl TDE1 (Illumina), 19 μl nuclease-free water) and incubated for 10 min at 37 °C.

After tagmentation, the beads were washed once with RIPA and once with cold Tris-Cl pH 8. Bead bound tagmented chromatin was resuspended in 10.5 μl 20 mM EDTA and incubated for 30 min at 50 °C. Then, 10.5 μl 20 mM MgCl₂ as well as 25 μl pre-activated 2x KAPA HiFi HotStart Ready Mix (incubation at 98 °C for 45 s, (Kapa Biosystems) were added and incubated for 5 min at 72 °C, followed by incubation for 10 min at 95 °C. Beads are magnetized and 2 μl of each library were amplified in a 10 μl qPCR reaction containing 0.8 mM primers, SYBR Green, and 5 μl Kapa HiFi HotStart ReadyMix to estimate the optimum number of enrichment cycles, using the following program: 72 °C for 5 min, 98 °C for 30 s, 24 cycles of 98 °C for 10 s, 63 °C for 30 s, 72 °C for 30 s, and a final elongation at 72 °C for 1 min. Kapa HiFi HotStart ReadyMix was incubated at 98 °C for 45 s before preparation of all PCR reactions (qPCR and final enrichment PCR), in order to activate the hot-start enzyme for successful nick translation at 72 °C in the first PCR step.

Final enrichment of the libraries was performed in a 50 μl reaction using 0.75 mM primers (custom Nextera primers as described for ATAC-seq) and 25 ml Kapa HiFi HotStart ReadyMix. Libraries were amplified for the for the number of cycles corresponding to the C_q value determined in the qPCR reaction. Enriched libraries were purified using SPRI AMPure XP beads. To prepare input control samples, 3μl of 50mM MgCl₂ was added to 15μl sonicated lysate (pool of 5μl of endothelium, epithelium, and fibroblasts lysates from the same organ) to neutralize the EDTA in the SDS lysis buffer; 20μl of tagmentation buffer and 1μl transposase (Illumina) was added, and samples were incubated at 37°C for 10min; chromatin was purified with MinElute PCR purification kit (Qiagen), and 22.5μl of the purified transposition reaction were combined with 25μl of PCR master mix and 0.75 mM primers (custom Nextera). Control libraries were amplified as described above. Libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq 3000/4000 platform and the 50-bp single-end configuration. Epigenome mapping by ChIPmentation was done in two biologically independent experiments, with two exceptions: for endothelium from lymph node and fibroblasts from thymus, only one high-quality profile could be obtained). Sequencing statistics are provided in Supplementary Table 1.