Neb monarch dna cleanup kit

ATAC-seq was performed as described previously (Lu et al. 2017 (link)). For each sample, approximately 1 g flash-frozen wheat samples were chopped with a razor blade in 1 mL ice-prechilled lysis buffer (15 mM Tris–HCl pH 7.5, 20 mM NaCl, 80 mM KCl, 0.5 mM spermine, 5 mM 2-Mercaptoethanol, 0.2% TritonX-100). The chopped slurry containing crude nuclei extract was filtered twice through a 40 μm filter. The crude nuclei were stained with DAPI (sigma, catalog number: D9542) and loaded to a flow cytometer (BD FACSCanto) for selected. Nuclei pellets were obtained after centrifuged and washed with Tris-Mg buffer (10 mM Tris–HCl pH 8.0, 5 mM MgCl₂). The obtained nuclei were incubated with 3.5 μL Tn5 transposomes in 40 μL TTBL buffer (TruePrep DNA Library Prep Kit V2 for Illumina, Vazyme Biotech co., ltd, TD501) for each sample at 37 °C for 30 min without rotation. We purified the integration products with a NEB Monarch™ DNA Cleanup Kit (T1030S) and then amplified for 10–13 cycles using the NEBNext Ultra II Q5 master mix (M0544L). PCR cycles were determined as described previously (Lu et al. 2017 (link)). Amplified libraries were purified with Hieff NGS® DNA Selection Beads (Yeasen, 12601ES03) to remove free index primers.

OCI-Ly7 cells were subjected to CUT&RUN assay according to the EpiCypher CUTANA CUT&RUN Protocol⁷⁹ . In brief, 500 K cells were immobilized onto activated ConA magnetic beads (Bangs Laboratories, BP531), followed by permeabilization and incubation with 0.5 ug of antibodies (H3K4me1, 13-0040; H3K4me3, 13-0028; or H3K27ac, 13-0045; SNAP-Certified for CUT&RUN and ChIP by EpiCypher) overnight at 4 °C. On the next day, the cell-bead slurry was washed and incubated with pAG-MNase (1:20 dilution, EpiCypher, 15-1116) for 10 min at RT. MNase was then activated by addition of CaCl2 to cleave targeted chromatin for 2 h at 4 °C. After chromatin digestion, MNase activity was stopped and chromatin fragments released into supernatant were purified using the NEB Monarch DNA Cleanup Kit (NEB, T1030) per manufacturer’s instruction. 10 ng DNA was subjected to library preparation using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645) according to the manufacturer’s protocol. Libraries were loaded onto the Illumina HiSeq for pair-end 150 bp sequencing with a sequencing depth of around 6 M reads per sample.