title: GSM7111552: Med1_2hLPS_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: For MED1 ChIP, 100x106 RAW264.7 cells were fixed with a double cross-linking protocol as previously described (Milan et al., 2019). Briefly, cells were scraped in PBS, incubated for 45 min with 2 mM DSG in PBS, washed twice, and then incubated with 1% formaldehyde in PBS for 10 min followed by quenching with 125 mM glycine. After chromatin shearing by sonication with a Branson Ultrasonics Sonifier, lysates were incubated overnight at 4°C with protein G Dynabeads (cat. no.10009D, Invitrogen) previously coupled with 10ug of MED1 antibody (cat.no. A300-793A, Bethyl). After immunoprecipitation, beads were recovered using a magnetic stand and washed with RIPA buffer (50 mM HEPES-KOH at pH 7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-deoxycho- late). Immunoprecipitated chromatin was eluted and cross-link-reverted overnight at 65°C. DNA was purified with QIAquick PCR purification kit (cat. no. 28106, Qiagen) and then quantified with QuantiFluor (cat. no. E2670, Promega). DNA libraries were prepared as previously described (Austenaa et al., 2021). The purified DNA libraries were quantified with the Quantifluor reagent (cat. no. E2670, Promega) and the quality of the size was controlled with the Tapestation instrument (Agilent) using the high-sensitivity assay HD5000 (cat. no. 5067-5592, Agilent). DNA libraries were diluted to a working concentration of 4 nM and 50-bp single reads sequenced on an Illumina Novaseq6000 platform.