Protein a coated beads

For the recombinant human CLEC-2–6xHis tag and human podoplanin-rabbit Fc vector overexpression, the extracellular domain of the cDNA of human CLEC-2 and podoplanin were cloned into a pHLSEC (Addgene) and pFUSE (Invivogen) expression vectors. The constructs were used to transient transfection into human embryonic kidney HEK293T cells. Cells were grown in complete Dulbecco's modified eagle medium (DMEM; supplemented with 10% fetal bovine serum, glutamine 5%, and 100 U/mL of penicillin/streptomycin). Once 60% of cell confluence was achieved, the medium was exchanged for serum-free DMEM and transfected using the PEI method. Proteins secreted into the medium and were collected after 4 days. hCLEC-2–6xHis tag and h-podoplanin-rFc were purified using a Nickel-NTA Resin (Thermo Fisher) and protein A-coated beads (Thermo Fisher), respectively, in a gravity purification column. SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis) was used to characterize proteins. Protein coding sequences of recombinant proteins can be found in the Supplementary Information.

A total of 20 million MyPL1 and MyPL2 cells were cross‐linked with 1% formaldehyde (Sigma‐Aldrich, F1635) for 10 min at room temperature, followed by a 5‐min treatment with 0.125 M glycine (Sigma‐Aldrich, G‐8790). Nuclei were isolated and chromatin was purified by chemical lysis. The purified chromatin was fragmented into 200–300 bp fragments by sonication (Covaris, S220) using the truChIP chromatin shearing kit (Covaris). Chromatin immunoprecipitation was performed by incubating the chromatin fraction overnight with 100 μL of protein‐A coated beads (Thermo‐Scientific, 53,139) and 8 μg of the H3K27Ac antibody (Abcam, ab4729). The next day, beads were washed to remove nonspecific binding events and enriched chromatin fragments were eluted from the beads, followed by reverse cross‐linking by incubation at 65°C overnight. DNA was subsequently purified by phenol/chloroform extraction, assisted by phase lock gel tubes (5Prime). DNA obtained from the ChIP assays was adaptor‐ligated, amplified, and analyzed by Illumina sequencing. Raw sequencing data were mapped to the mouse reference genome (GRCm38) using Bowtie. Peak calling was performed using MACS2.
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