Instrument: Illumina HiSeq 4000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Organs were digested with Accumax and structural cells isolated using FACS ChIPmentation was carried out as previously described (Gustafsson et al., 2019; Schmidl et al., 2015), with minor adaptions. Briefly, 50,000 cells (or a lower number of cells for particularly rare cell populations) were washed once with PBS. Cells were fixed and sonicated (Covaris S220) as described (Schmidl et al., 2015). Following sonication, the lysate was brought 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) and 1 mM PMSF) to a volume of 200 µl per immunoprecipitation. For each immunoprecipitation, 10 µl magnetic Protein A (Life Technologies) were washed twice and resus-pended in PBS supplemented with 0.1% BSA. 1 µg of antibody recognizing H3K4me2 (MerckMillipore, clone AW30) was added and bound to the beads by rotating over night 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 and tagmenting the bead-bound chromatin as described (Gustafsson et al., 2019). After tagmentation, the beads were washed once with RIPA and once with cold Tris-Cl pH 8. Bead bound tagmented chromatin was than 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 of pre-activated 2x KAPA HiFi HotStart Ready Mix (incubation at 98 °C for 45 s) are 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 with 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 and 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), 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 rounded-down Cq value determined in the qPCR reaction. Enriched libraries were purified using SPRI AMPure XP beads. For preparation of compatible input controls, 3 μl of 50 mM MgCl2 was added to 15 μl sonicated lysate (pool of 5 μl of endothelium, epithelium and fibroblasts lysates form the same organ) to neutralize the EDTA in the SDS lysis buffer. 20 μl of tagmentation buffer and 1 μl transposase (Nextera, Illumina) was added, and samples were incubated at 37 °C for 10 min. Input control chromatin was purified with MinElute PCR purification kit (Qiagen) and 22.5 μl of the purified transposition reaction were combined with 25 μl of PCR master mix and 0.75 mM primers (custom Nextera). Libraries were subsequently amplified as described above. The libraries were sequenced by the Biomedical Sequencing Facility at CeMM using the Illumina HiSeq3000/4000 platform and the 50-bp single-end configuration