Open Chromatin Labeling and NicE-seq library construction using Nt.CviPII
Open Chromatin Labeling
- Harvest cells and count to determine the cell density using automated cell counter or hemocytometer.
- Dilute the sample to obtain the desired number of cells in the tube.
- Crosslink the cells using 1% formaldehyde for 10 min at room temperature, followed by quenching the fixation using 125 mM Glycine.
- Wash the cells using 1X PBS to remove residual formaldehyde.
- Isolate nuclei by resuspending cells in cytosol extraction buffer (15 mM Tris-HCl pH 7.5, 5 mM MgCl2, 60 mM KCl, 0.5 mM DTT, 15 mM NaCl, 300 mM sucrose, and 1% NP40) and incubating on ice for 10 min with occasional agitation.
- Precipitate the nuclei by spinning at 1000 X g, 4˚C for 5 minutes and discard the supernatant.
- Label open chromatin by incubating the nuclei in 2.5 U of Nt.CviPII (NEB #R0626S), 10 U of DNA Polymerase I (NEB #M0209S) and 30 μM of each dNTP including 6 μM of biotin-14-dATP (Invitrogen, 19524-016) and 6 μM of biotin-16-dCTP (ChemCyte, CC-6003-1) in 200 μl of 1X NEBuffer 2 at 37˚C for 2 hours.
- Stop the labeling reaction by adding 20 µl of 0.5 M EDTA and 2 µg of RNase A to the reaction and incubate for 30 min at 37˚C.
- De-crosslink the DNA-protein crosslinking by adding 20 µl of Proteinase K (NEB #P8107S) and 20 µl of 20% SDS to the reaction and incubating overnight at 65˚C.
- Extract biotin labeled genomic DNA using phenol chloroform method.
NicE-seq library construction
- Fragment the biotin labeled genomic DNA into 150 bp fragments using Covaris®.
- Generate libraries with fragmented DNA (up to 1 µg) using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645S).
- Following adaptor ligation, without further purification, add 50 µl of streptavidin magnetic beads (blocked with 0.1% cold fish gelatin at 4˚C) to the library, resuspended in 1 ml of B&W buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 2 M NaCl) and incubate at 4˚C for 2 hours with end-over-end mixing.
- Wash the streptavidin bead bound library four times on the magnet using B&W buffer supplemented with 0.05% Triton X-100 and once with 1X TE plus 0.05% Triton X-100.
- Finally, resuspend the beads in 40 µl of nuclease-free water and use 4 µl for library amplification as per the guidelines given in NEBNext Ultra II DNA Library Prep Kit.