Neb ultra fs 2 dna library kit

Sample collection and Sequencing: CUT&RUN was performed as described 13 (link) . Briefly, Nthy-ORI cells were harvested, washed, and bound to activated Concanavalin A coated magnetic beads (Epicypher 21-1401). Cells were then permeabilized with Wash buffer (20mM HEPES pH 7.5, 150mM NaCl, 0.5 mM spermidine 0.05% digitonin). Permeabilized cells were then incubated with the indicated antibody (Supplemental Table 3) at 4°C with constant agitation overnight. Cells were washed twice more before incubation with recombinant p-AG MNase (Epicypher 15-1016) at 4°C for 2 hours. Liberated DNA was purified, and libraries were prepared using the NEB Ultra FS II DNA Library Kit (NEB E6177) and amplified with 14 cycles of PCR. Amplified libraries were then purified with AMPure beads (Agencourt), quantified via Qubit (Life Technologies), and quality was assessed using the BioAnalyzer (Agilent) High-Sensitivity DNA kit. CUT&RUN libraries were pooled and sequenced on the Illumina HiSeq 1500 with 100 bp paired-end reads.
Data Analysis: Quality scores across sequenced reads were assessed using FASTQC. Illumina adapters were removed using Trim-Galore. Paired-end reads were mapped to hg38 using Bowtie2, and peaks were called using MACS2. Consensus peak sets for downstream analysis were derived using IDR 29 using two replicates (Supplementary Figure 2A) per target and a cutoff of 0.05.