GSM7914671: Ptch1, H3K4me3, Control, CUT&RUN, rep2; Mus musculus; OTHER
Cut&Run assays were performed with CUTANATM ChIC/CUT&RUN kit according to the manufacturer's protocol (EpiCypher, #14-1048). Briefly, fresh 500,000 cells were collected with a cell lifter (Corning, #3008) and resuspended in wash buffer (Pre-wash buffer (EpiCypher, #21-1002), protease inhibitor, and 0.5 mM spermidine and bound to activated Concanavalin A beads. Cells conjugated with beads were incubated with Antibody buffer (Cell permeabilization buffer and 2 mM EDTA) and antibodies at 4°C overnight. After extensive washing with Cell permeabilization buffer (Wash buffer and 0.01% digitonin), the cells were incubated with pAG-MNase (EpiCypher, #15-1016) for 10 min at room temperature, and then chromatin digestion was performed with calcium chloride for 2 h at 4°C. SNAP CUTANA™ Spike-in Controls for CUT&RUN (Cat: SKU-19-1002) panels of modified DNA-barcoded Designer Nucleosomes (dNucs™) carrying an array of widely studied histone post-translational modifications (PTMs) were used. After chromatin digestion, the stop buffer and E. coli spike-in DNA (EpiCypher, #18-1401) were added and incubated for 10 min at 37°C. Chromatin fragments were purified with DNA cleanup columns. Library preparation on samples was conducted using manufacturer's protocols for NEBNext Ultra II Dna Library Prep Kit (New England BioLabs, CAT#E7645) and NEB Multiplex Dual Index oligos (New England BioLabs, CAT#E7600, #E7780) with the following modifications. We input approximately 10ng of sample for half reactions, we diluted the NEBNext Illumina adaptor 1:25, we used the following PCR cycling conditions: 1 cycle of Initial Denaturation at 98C for 30 seconds, 12+ cycles of Denaturation at 98C for 10 seconds then Annealing/Extension at 65C for 10 seconds, and 1 final cycle of extension at 65C for 5 minutes. Following library preparation, double size selection was performed using Ampure beads and quality and concentration of libraries were determined by an Agilent 4200 Tapestation with High-Sensitivity D1000 reagents before pooling for sequencing.