Nebnext ultra 2 dna library prep kit

CUT&RUN analysis was performed with approximately 2 × 10⁵ naive ESCs for each library. Bead-bound cells were incubated overnight at 4 ^oC with Rabbit anti-H3K4me3 (Millipore, #07-473) or Rabbit anti-H3K27me3 (Millipore, #07-449) antibody diluted in the antibody buffer (EDTA/Digitonin/wash buffer, EMD Millipore, #300410) (1 µg/500 µl). The beads were washed three times and incubated with 200 µl pA-MNase (0.7 ng/µl) for 1 h at 4 ^oC. 2 mM of CaCl₂ was added to activate MNase for 30 min on ice. The reaction was quenched by 2× stop buffer containing 340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.02% Digitonin, 50 µg/ml RNase A (QIAGEN, #19101), 50 µg/ml glycogen (Roche, #10901393001) and 2 pg/ml Drosophila spike-in DNA at 37 ^oC for 10 min. The soluble chromatins were converted to libraries using the NEBNext Ultra^TM II DNA library Prep kit (NEB, #E7645L). Libraries were subject to pair-ed sequencing on the Illumina NovaSeq 6000 instrument at the University of Michigan core facility.

DNA was extracted from high titer lysates using the Wizard® Clean-Up Kit (cat. # A7280, Promega, WI, USA). Sequencing libraries were constructed with the NEBNext® UltraTM II DNA Library Prep kit (New England Biolabs, MA, USA), and shotgun sequenced by Illumina-MiSeq at the Pittsburgh Bacteriophage Institute. Genome assembly and finishing were conducted as previously described [19 ].

Bacteriophage whole genome sequencing was performed on the Illumina MiSeq^® technology using the NEBNext^® Ultra^TM II DNA Library Prep Kit (NEB) and a MiSeq^® V3 600 cycle reagent kit (Illumina, Australia) according to manufacturer’s instructions. Generated reads were trimmed using Trim Galore v0.6.4 with the default settings (Q scores of ≥ 20, with automatic adapter detection) and assembled using the Unicycler de novo assembly pipeline (Wick et al., 2017 (link)). PhageTerm was used to reorient the phage genome to begin at its pac site (Garneau et al., 2017 ) before exporting to Geneious (Version 11.0.5)¹. Translated open reading frames (ORFs) were mapped onto the National Centre for Biotechnology Institute (NCBI) database using BLASTP (Mount, 2007 ) and annotated sequence submitted to GenBank^®. To allocate taxa for bacteriophage EFA1, Viral Proteomic Tree (ViPTree) webserver was used to construct a viral proteomic tree with other related bacterial viral genomes in the reference database (Nishimura et al., 2017 (link)). The generated proteomic tree was annotated using the Interactive Tree of Life (iTOL) (Letunic and Bork, 2019 (link)). Amino acids of putative beta-lactamase protein in bacteriophage EFA1 and bacteria (E. faecalis) were aligned by a pair-wise progressive alignment in CLC genomics workbench version 9.5.4.

Probes were ordered as ssDNA molecules from IDT (Supplementary Table S3). Constant sequences included a 5′ P5 Illumina adaptor, a nucleotide stagger ranging from 1 to 8 bp, two discrete 6 bp anchoring sequences, a fully randomized barcode for multiplexed sequencing reactions, a UMI probe (UMI_p) to control for PCR bias and identify unique ligation events, and a constant 3′ anchoring sequence to facilitate probe amplification.
Probes were PCR amplified prior to use. All PCR reactions were prepared with 25 μL 2× NEBNext Ultra II DNA Library Prep Kit (NEB E7645S), 0.5 μM of both P5 adaptor and Primer_P primers, and 0.18 μL 100 μM probe. Reactions were run at 95°C for 1 min, 20 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 7 min. The PCR product was concentrated with the Zymo DNA Clean & Concentrator Kit (Zymo D4004) and normalized to 30 ng/μL.
All ligation reactions were prepared using 5 μL of digested template library, 3 μL of 30 ng/μL probe, 1 μL 10× T4 Ligase buffer, and 1 μL high concentration T4 Ligase (NEB M0202M). Ligation reactions were performed at room temperature for 1 h.

Template libraries received from IDT were brought to 100 μM in low TE (10 mM Tris-HCL, 0.1 mM EDTA, pH 8.0) and then serially diluted 1,000-fold in 10× increments. Concentrations of each dilution were determined with the Qubit ssDNA Assay Kit (Qubit Q10212) and diluted to 250,000 copies/μL. Libraries were polymerase chain reaction (PCR) amplified with approximately 62,500 template copies per reaction across eight technical replicates, or 500,000 templates total. All PCR reactions were prepared with 25 μL 2× NEBNext Ultra II DNA Library Prep Kit (NEB E7645S) and 0.5 μM of P7-Adaptor and Primer_T primers. Reactions were run at 95°C for 1 min, 27 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 7 min. The PCR product was concentrated with the Zymo DNA Clean & Concentrator Kit (Zymo D4004) to facilitate downstream reaction preparation.

CTCF ChIP was conducted using Millipore ChIP agarose kit (Millipore). 1 × 10⁶ cross-linked 416B cells were lysed and sonicated using a Covaris ultrasonicator. Sonicated chromatin was diluted using dilution buffer and 50 µL was removed as the 5% input control. 2 µL CTCF antibody (Supplementary Table 3) was added to 1 mL chromatin and incubated overnight at 4 °C. Decross-linking was done at 65 °C overnight. DNA was purified using phenol-chloroform-isoamylalcohol (25:24:1, Sigma) and enrichment was determined using qPCR (Supplementary Table 5). NEBNext Ultra II DNA Library Prep Kit (NEB) with 11 cycles of PCR was used to prepare sequencing libraries. CTCF ChIP libraries were sequenced using Illumina NextSeq high-output 75 cycle kit (paired-end).

RNA-seq libraries were prepared as previously described. Briefly, three embryos were used per reaction, and two replicates were performed for each group. All embryos were washed three times in 0.5% BSA-PBS solution to avoid possible contamination. cDNA was amplified using the Phusion Hot Start II High-Fidelity PCR Master Mix (F-565S; Thermo Fisher). Library preparation was performed using the NEBNext Ultra II DNA Library Prep Kit (E7805S, New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. All libraries were sequenced using the NovaSeq 6000 (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions.