Bead Activation Buffer (pH with KOH) pH HEPES to 7.9 before adding salts
20 mM HEPES, pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2
Filter sterilize Store @ 4ºC for up to 6 months
Wash Buffer (pH with KOH)
20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine
Store @ 4ºC for up to 1 week
1x Roche Complete Protease Inhibitor-mini (CPI-mini), EDTA-free (Roche catalog # 11836170001), 1tab/10ml, added fresh before use
Dissolve 5 mg in 100 ul DMSO.Store @ 4ºC.Good for one week.
Wash Buffer + 0.01% Digitonin *
* Optimal [Digitonin] for each cell type should be empirically determined. Starting concentration validated for K562, MCF7, and A549 cells = 0.01% digitonin.
Prepare fresh each day and store @ 4ºC
Wash Buffer + 0.01% Digitonin + 2 mM EDTA
Prepare fresh each day and store @ 4ºC
Filter sterilize. Store @ 4ºC for up to 6 months
*ADD 150 pg/150,000 cells of Epicypher’s E. coli spike-in (optional)
IgG antibody, Cell Signaling, Cat# 2729
H3K27ac antibody, Cell Signaling, Cat# 8173
H3K27me3 antibody, Cell Signaling, Cat# 9733
H3K4me1 antibody, Cell Signaling, Cat# 5326
CUTANA Concanavalin A magnetic beads, Epicypher, Cat# 21-1401
CUTANA pAG-MNase, Epicypher, Cat# 15-1016
CUTANA E. coli Spike-in DNA, Epicypher, Cat# 18-1401
CUTANA DNA purification kit, Epicypher, Cat# 14-0050
NEBNext® UltraTM II Library Prep Kit for Illumina®, NEB, Cat# E7645
NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs), NEB, Cat# E6440S
Digitonin, Millipore Sigma, Cat# 300410
RNase, Sigma Millipore, Cat# R4642
Glycogen, Roche, Cat# 10901393001
Spermidine, Sigma Millipore, Cat# S2501
cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail, Roche, Cat# 11836170001
Molecular biology grade water (RNase, DNase free)
EDTA (0.5M stock, pH 8.0)
EGTA (0.5M stock, pH 8.0)
Fisherbrand RNase-Free Disposable Pellet Pestles, Fischer Scientific, Cat# 12-141-364
CellTrics 30 µm Filter, Sysmex, Cat# 04-004-2323
GeneMate 1.7 mL Low-Adhesion tubes, VWR, Cat# 490003-230
1.5 mL Magnetic Separation Rack