title: GSM6564359: Formative_ESC_WT_MLL4_rep1; Mus musculus; OTHER
library_name: GSM6564359
library_construction_protocol: CUT&RUN was conducted using the protocol from Skene et al. 2018 with the following modifications: Freshly trypsinized cells were bound to activated Concanavalin A beads (Bang Laboratories, CAT#BP531) at a ratio of 2e5 cells/10ul beads in CR Wash buffer (20mM HEPES, 150mM NaCl, 0.5mM Spermidine with protease inhibitors at room temp. An input of 2e5 cells were used per target. Bead-bound cells were then incubated rotating overnight at 4C in CR Antibody buffer (CR Wash with 0.05% Digitonin, 2mM EDTA) containing primary antibody. We used the following antibodies for CUT&RUN: 1:100 Rabbit anti-H3K4m1 (Abcam, ab4729), Rabbit anti-MLL4 1:100 (provided by Kai Ge), and 1:100 Rabbit IgG isotype control (Abcam, ab171870). After primary, we washed 3 times 5 minutes each with cold CR Dig-wash buffer (CR Wash with 0.05% Digitonin) and incubated with pA-MNase (1:100 of 143ug/mL provided by Steve Henikoff) for 1 hour rotating at 4C. After MNase binding, we washed 3 times 5 each with cold CR Dig-wash buffer, and chilled cells down to 0C using a metal tube rack partially submerged in an ice water slurry. MNase digestion was induced by adding CaCl2 at a final concentration of 2mM. After 30 minutes of digestion, the reaction was quenched using Stop Buffer containing 340mM NaCl, 20mM EDTA, 4mM EGTA, 0.05% Digitonin, 100ug/mL RNAse A, 50ug/mL Glycogen, and approximately 2pg/mL Yeast spike-in DNA (provided by Steve Henikoff). The digested fragments for each sample were then extracted using a phenol chloroform extraction. 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.