GSM8447828: lungs, Control, rep1; Mus musculus; OTHER
Lungs from E17.5 Wild Type C57Bl6 mice were harvested, minced, and incubated in RPMI media containing Liberase TM (Roche, 50ug/ml) and DNAse I (MilliporeSigma, 100ug/ml) at 37C for 30min. Tissues were dissociated with GentleMACS cell dissociator, then subjected to RBC lysis buffer for 2min on ice, followed by an incubation with FcR blocking reagent (Miltenyi: 130-092-575) on ice for 10min, followed by an incubation with CD326 anti-Mouse-EpCAM microbeads (Miltenyi: 130-105-958) at 4C for 15min. Cells were subjected to positive selection with an LS column and collected in MACS buffer (Miltenyi). Cells were quantified manually via hemacytometer with trypan blue. 100,000 EpCAM+ cells were bound to concanavalin A beads, then incubed with antibodies against H3K4me3. IgG controls with either a rabbit or sheep IgG were also generated. Cut&Run Assay Kit (Cell Signaling Technologies, 86652S) was used. Cells were incubated with pAG-Mnase to facilitate anitbody-guided DNA digestion. The extracted DNA was purified by phenol-chloroform-isoamyl alcohol (Invitrogen: 15593-031) extraction and  Phasemaker tubes (Invitrogen: A33248) DNA was quantified using a Qubit (ThermoFisher) and libraries were constructed using the Illumina DNA Library Prep Kit (Cell Signaling Technology, 56795S) and Multiplexoligos (47538S). Libraries were sequenced for at least 15mil reads. Libraries sequenced by Genewiz