Sfx250 digital sonifier

ChIP was performed as described previously^{72 (link)} with some modifications. In brief, HA-tagged MafB overexpressed MS1 ECs were cross-linked with 1% (wt/vol) formaldehyde for 10 min at room temperature and then added glycine to a final concentration of 0.15 M. Chromatin was sonicated to an average fragment length of 150–250 bp using SFX250 digital sonifier (Branson) for 2 min: 0.7 s on/1.3 s off on ice and immunoprecipitated with anti-HA antibody-conjugated magnetic beads (M180-11; MBL) overnight at 4 °C. Following protein–DNA complex elution, samples were treated with RNaseA and proteinase K and incubated at 65 °C overnight for reverse cross-linking. Immunoprecipitated DNA was purified using AMPure XP SPRI beads (Beckman Coulter). Sequencing libraries were prepared with NEBNext Ultra DNA Library Prep Kit for Illumina (New England BioLabs) according to the manufacturer’s instructions and sequenced on a MiSeq sequencer using 2 × 75 bp paired-end MiSeq v3 chemistry (Illumina). Sequencing reads were aligned to the mm10 reference mouse genome using Bowtie2^{73 (link)} and analyzed with HOMER^{31 (link)} for peak finding, motif analysis and peaks annotation. Gene set annotation enrichment tests were performed using GREAT v.3.0^{32 (link)} with default parameters and were subsequently used for calculating the overlap with DEGs in the Mafb-cKO RNA-Seq experiment.

Human cervix carcinoma (HeLa) cells were cultured in Dulbecco’s modified Eagle's medium (Life Technologies Ltd) containing 20 mM glutamine, 10% fetal bovine serum, and 1% penicillin–streptomycin. After harvest, the cells were resuspended in PreOmics lysis buffer and incubated at 95 °C for 10 min to reduce disulfide bridges, alkylate cysteine residues, and denature proteins. Samples were sonicated using a rod sonicator (Branson SFX 250 Digital Sonifier) and subsequently incubated at 95 °C for an additional 5 min. HeLa cell lysates were diluted with an equal volume of water and digested overnight using equal amounts of LysC and trypsin (1:100 ratio at protein level). Following digestion, peptides were acidified to a final concentration of 1% TFA and purified on StrataTM-X-C (Polymeric Strong Cation) cartridges. Peptides were eluted in 80% acetonitrile (ACN)/1.25% NH₄OH and subsequently dried using a SpeedVac (Eppendorf). Samples were resuspended in buffer A∗ (0.1% TFA, 2% ACN, or buffer A [0.1% formic acid (FA)]), for measurement with the Thermo Scientific EASY-nLC 1200 system or the Evosep LC system, respectively. Peptide concentrations were estimated by measuring absorbance at 280 nm on a Thermo Scientific NanoDrop 2000 spectrophotometer. For online MS injection using the Evosep One (LC) system, peptides were loaded onto Evotips according to the manufacturer’s instructions.