A Dovetail Chicago library was prepared following established procedures.18 (link) Briefly, ~500 ng of high molecular weight gDNA was reconstituted into chromatin in vitro and fixed with formaldehyde. Fixed chromatin was digested with DpnII, the 5ʹ overhangs filled in with biotinylated nucleotides, and then free blunt ends were ligated. After ligation, crosslinks were reversed and the DNA purified from protein. Purified DNA was treated to remove biotin that was not internal to ligated fragments. The DNA was then sheared to ~350 bp mean fragment size and sequencing libraries were generated using NEBNext Ultra enzymes and Illumina-compatible adapters. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment of each library. The libraries were sequenced on an Illumina HiSeq X to produce 108 million 2 × 150 bp paired end reads.
Nebnext ultra enzymes
Manufactured by Illumina
Sourced in United States
The NEBNext Ultra enzymes are a set of molecular biology reagents designed for applications such as library preparation for next-generation sequencing. They include a collection of enzymes, adaptors, and buffers optimized for efficient and reliable DNA/RNA processing.
Automatically generated - may contain errors
12 protocols using nebnext ultra enzymes
1
Dovetail Chromatin Conformation Capture
2
In Vitro Chromatin Reconstitution and Hi-C Sequencing
3
De novo genome sequencing of Hermetia illucens
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All de novo genome sequencing was carried out by Dovetail Genomics. High-molecular-weight DNA was extracted from 3 H. illucens degutted males, and a PacBio circular consensus sequencing (CCS) library was produced and sequenced on the PacBio™ HiFi™ platform (PacBio, San Diego, CA, USA). A Dovetail Omni-C library was also prepared. Chromatin was fixed in place with formaldehyde in the nuclei of an H. illucens adult male and then extracted. Fixed chromatin was digested with DNAse I, and chromatin ends were repaired and ligated to a biotinylated bridge adapter followed by proximity ligation of adapter-containing ends. After proximity ligation, crosslinks were reversed, and the DNA was purified. The purified DNA was treated to remove biotin that was not internal to ligated fragments. Sequencing libraries were generated using NEBNext Ultra enzymes and Illumina-compatible adapters (New England Biolabs, Ipswich, MA, USA). Biotin-containing fragments were isolated using streptavidin beads before the PCR enrichment of each library. The Dovetail Omni-C library was then sequenced on an Illumina HiSeqX platform (Illumina, San Diego, CA, USA).
4
Dovetail Hi-C Library Preparation
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A Dovetail Hi-C library was prepared in a similar manner as described in Lieberman-Aiden et al. (2009) (link). For each library, chromatin was fixed with formaldehyde in the nucleus and then extracted. This was then digested with DpnII and the 5′ overhangs were filled with biotinylated nucleotides. After the free blunt ends were ligated, crosslinks were reversed and the DNA was purified from protein. Biotin that was not internal to ligated fragments was removed and DNA was then sheared to a mean fragment size of 350 bp. Sequencing libraries were generated using NEBNext Ultra enzymes and Illumina-compatible adapters. Biotin-containing fragments were isolated using streptavidin beads before the PCR enrichment of each library. Finally, the libraries were sequenced on an Illumina HiSeq X to produce 200 million 2x150 bp paired-end reads.
5
Hi-C Protocol for Genome Mapping
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A Chicago library was prepared as described previously (Putnam et al., 2016 ). Briefly, ~500 ng of HMW gDNA (mean fragment length = 85 kbp) was reconstituted into chromatin in vitro and fixed with formaldehyde. Fixed chromatin was digested with DpnII, the 5′ overhangs filled in with biotinylated nucleotides, and free blunt ends were ligated. After ligation, crosslinks were reversed and the DNA purified from protein. Purified DNA was treated to remove biotin that was not internal to ligated fragments. The DNA was then sheared to ~350 bp mean fragment size and sequencing libraries were generated using NEBNext Ultra enzymes and Illumina‐compatible adapters. Biotin‐containing fragments were isolated using streptavidin beads before PCR enrichment of each library. The libraries were sequenced on an Illumina HiSeq X to produce 211 million 2 × 150 bp paired end reads, which provided 146.61 × physical coverage of the genome (1–100 kb pairs).
6
Hi-C Library Preparation Protocol
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The Hi-C library was generated by Dovetail Genomics as described by Lieberman-Aiden et al. (2009) (link). Briefly, for each library, chromatin was fixed in place with formaldehyde in the nucleus and then extracted. Fixed chromatin was digested with DpnII, the 5′ overhangs filled in with biotinylated nucleotides, and then free blunt ends were ligated. After ligation, crosslinks were reversed, and the DNA purified from protein. Purified DNA was treated to remove biotin that was not linked to ligated fragments. The DNA was then sheared to a mean fragment size of ∼350 bp, and sequencing libraries were generated using NEBNext Ultra enzymes and Illumina-compatible adapters. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment of each library. The libraries were sequenced on an Illumina Hi-seq 4000 sequencing platform (2 × 151 bp).
7
Hi-C Library Preparation Protocol
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Three Dovetail Hi-C libraries were prepared for each sample in a similar manner as described previously [39 (link)]. Briefly, for each library, chromatin was fixed in place with formaldehyde in the nucleus and then extracted. Fixed chromatin was digested with DpnII, the 5’ overhangs were filled in with biotinylated nucleotides, and then the free blunt ends were ligated. After ligation, crosslinks were reversed, and the DNA was purified from protein. Purified DNA was treated to remove biotin that was not internal to ligated fragments. The DNA was then sheared to ~350 bp mean fragment size and sequencing libraries were generated using NEBNext Ultra enzymes and Illumina-compatible adapters. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment of each library.
8
Omni-C Chromatin Proximity Ligation
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Proximity ligation and sequencing was conducted by Dovetail using Omni-C sequencing for both species. For each Dovetail Omni-C library, chromatin was fixed in place with formaldehyde in the nucleus and then extracted. Fixed chromatin was digested with DNAse I, and chromatin ends were repaired and ligated to a biotinylated bridge adapter followed by proximity ligation of adapter-containing ends. After proximity ligation, crosslinks were reversed, and the DNA was purified. Purified DNA was treated to remove biotin that was not internal to ligated fragments. Sequencing libraries were generated using NEBNext Ultra enzymes and Illumina-compatible adapters. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment of each library. The libraries were sequenced on an Illumina HiSeqX platform to produce an approximately 30× sequence coverage.
9
Hi-C Sequencing Library Preparation
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DNA was isolated as per an established method (Furtado, 2014 ). Then, the library was prepared as described in Putnam et al. (2016 (link)). Briefly, ~500 ng of HMW gDNA was reconstituted into chromatin in vitro and fixed with formaldehyde. Fixed chromatin was digested with DpnII, the 5′ overhangs filled in with biotinylated nucleotides, and then, free blunt ends were ligated. After ligation, crosslinks were reversed, and the DNA was purified from protein. Purified DNA was treated to remove biotin that was not internal to ligated fragments. The DNA was then sheared to ~350 bp mean fragment size, and sequencing libraries were generated using NEBNext Ultra enzymes and Illumina‐compatible adapters. Biotin‐containing fragments were isolated using streptavidin beads before PCR enrichment of each library. The libraries were sequenced on an Illumina HiSeqX platform to produce 213 million 2 × 150 bp paired end reads, which provided 88.11 × physical coverage of the genome (1–100 kb pairs).
10
Dovetail HiC Library Preparation
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3 Dovetail HiC libraries were prepared as described previously [13 (link)]. Briefly, for each library, chromatin was fixed in place with formaldehyde in the nucleus and then extracted. Fixed chromatin was digested with DpnII, the 5′ overhangs filled in with biotinylated nucleotides, and then free blunt ends were ligated. After ligation, crosslinks were reversed and the DNA purified from protein. Purified DNA was treated to remove biotin that was not internal to ligated fragments. The DNA was then sheared to a ∼350 bp mean fragment size, and sequencing libraries were generated using NEBNext Ultra enzymes and Illumina-compatible adapters. Biotin-containing fragments were isolated using streptavidin beads before PCR enrichment of each library. The libraries were sequenced on an Illumina Hi-seq 4000 sequencing platform (2 × 151 bp) (Illumina Hi-seq 4000, RRID:SCR_016386 ). The number and length of read pairs produced for each library were 141 million for library 1, 88 million for library 2, and 133 million for library 3. Together, these Dovetail HiC library reads provided 1990.23× physical coverage of the genome (1–50 kb pairs).
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