For each sample, 200 ng of sonicated fragments were used to make NGS (next-generation sequencing) libraries using NEBNext Ultra II DNA Library Prep (New England BioLabs, E7645S) in combination with methylated adaptors (NEB, E7535S), following the manufacturer’s instructions. Adaptor-ligated fragments were then purified with 1.0x Agencourt AMPure Beads (Beckman Coulter, Inc). Libraries were then treated with sodium bisulfite according to the manufacturer’s instructions (Imprint DNA Modification Kit; Sigma, MOD50) and amplified by PCR (10 cycles) using KAPA HiFi HS Uracil+ RM (KAPA Biosystems) and NEBNext Multiplex Oligos for Illumina (NEB E7335S). Bisulfite-converted libraries were finally size-selected and purified using 0.7x Agencourt AMPure Beads. The size and purity of libraries were determined using Tapestation and quantified using Qubit (Agilent). Whole-genome bisulfite sequencing (WGBS) libraries were sequenced on HiSeq 4000 (High Output mode, v.4 SBS chemistry) to generate paired-end 150 bp-long reads. A. stuartgranti samples were sequenced on HiSeq 2500 to generate paired-end 125 bp-long reads.
Nebnext ultra 2 dna library prep
Manufactured by New England Biolabs
Sourced in United States
The NEBNext Ultra II DNA Library Prep is a kit for preparing DNA samples for next-generation sequencing. It includes reagents and protocols for enzymatic DNA fragmentation, adapter ligation, and library amplification.
Automatically generated - may contain errors
Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Imprint dna modification kit, by Merck Group (1 mentions)
Agencourt ampure beads, by Beckman Coulter (1 mentions)
Mnase, by New England Biolabs (1 mentions)
Chip it high sensitivity kit, by Active Motif (1 mentions)
Puregene core kit a, by Qiagen (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Le220 focused ultrasonicator, by Covaris (1 mentions)
Pfuultra hf dna polymerase, by Agilent Technologies (1 mentions)
Sonicator, by Covaris (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Methylminer methylated dna enrichment kit, by Thermo Fisher Scientific (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
6 protocols using nebnext ultra 2 dna library prep
1
Whole-Genome Bisulfite Sequencing for DNA Methylation
2
ChIP-seq Protocol for p53 Enrichment
3
Whole Genome Sequencing and Somatic Variant Detection
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DNA was extracted by using the Puregene core Kit A (#1042601, Qiagen Inc., Germantown, MD). The DNA samples were fragmented using the Covaris LE220 Focused Ultrasonicator (Covaris, Valencia, CA), Fragmented genomic DNA from cell line samples were used as input for NEBNext Ultra II DNA Library Prep (NEB, Ipswitch, MA) and hybridized using Agilent SureSelect for Target Enrichment with the Agilent Exome Panel v7 (Agilent, Santa Clara, CA) according to the manufacturer’s instructions. Paired-end sequencing resulting in 150 bases from each end of the fragments for whole genome libraries was performed using Illumina NovaSeq 6000 instrumentation (Illumina, San Diego, CA). Human and mouse sequence reads were disambiguated using BBTools76 (link). Subsequent reads were aligned to the GRCh37 human reference genome using the Burrows-Wheeler Aligner v0.7.17-r118877 . Duplicate reads were marked using Samtools v78 (link). Somatic variants were called using Mutect2 following recommended filtering methods79 .
4
Mutation Analysis of IgH Sμ Region
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For the analysis of mutations at IgH Sμ region, genomic DNA was isolated from GC and naïve B cells from immunized mice. Amplifications were performed in 4 independent reactions using the following primers:
(forward) 5’- AATGGATACCTCAGTGGTTTTTAATGGTGGGTTTA-3’; (reverse) 5’- GCGGCCCGGCTCATTCCAGTTCATTACAG-3’. Amplification reactions were carried in a final volume of 25 μl each, using 2.5 U Pfu Ultra HF DNA polymerase (Agilent) for 26 cycles (94°C for 30”, 55°C for 30”, 72°C for 60”). PCR products were purified and fragmented using a sonicator (Covaris), and libraries were prepared according to the manufacturer’s instructions (NEBNext Ultra II DNA Library Prep; New England Biolabs). Sequencing was performed in a HiSeq 2500 platform (Illumina). Analysis was performed as previously described [30 (link)].
(forward) 5’- AATGGATACCTCAGTGGTTTTTAATGGTGGGTTTA-3’; (reverse) 5’- GCGGCCCGGCTCATTCCAGTTCATTACAG-3’. Amplification reactions were carried in a final volume of 25 μl each, using 2.5 U Pfu Ultra HF DNA polymerase (Agilent) for 26 cycles (94°C for 30”, 55°C for 30”, 72°C for 60”). PCR products were purified and fragmented using a sonicator (Covaris), and libraries were prepared according to the manufacturer’s instructions (NEBNext Ultra II DNA Library Prep; New England Biolabs). Sequencing was performed in a HiSeq 2500 platform (Illumina). Analysis was performed as previously described [30 (link)].
5
Whole Genome Sequencing of Metagenomic Samples
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The preparation of DNA libraries and whole genome sequencing were performed in Novogene Co., Ltd. (Hong Kong) with requested 100 Gb data output per each sample. Briefly, after quality control of extracted DNA, 300 bp fragments were prepared by sonication and the DNA libraries were constructed using NEBNext® Ultra™ II DNA Library Prep (New England Biolabs, USA). The libraries were diluted to 2 ng/μL and their quality (>3 nM) was verified by qPCR. The libraries were sequenced using HiSeq platform (Illumina, USA) with paired-end strategy (150 bp).
On average, metagenomic sequencing resulted in 365,613,222 reads and 109.69×109 bases per sample (Table S2
The data are available under BioProject ID: PRJNA666684 (NCBI database;https://dataview.ncbi.nlm.nih.gov/object/PRJNA666684 ).
On average, metagenomic sequencing resulted in 365,613,222 reads and 109.69×109 bases per sample (
The data are available under BioProject ID: PRJNA666684 (NCBI database;
6
Methylated DNA Enrichment and Sequencing
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Whole blood was collected into EDTA tubes, and DNA was extracted using a by DNeasy Blood & Tissue Kit (Qiagen Pty Ltd, Hilden, Germany) and quantified by Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). DNA was fragmented by sonication. The MethylMiner Methylated DNA Enrichment Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to enrich methylated DNA fragments from 500 ng of input material. DNA was eluted from beads with 2 M NaCl solution and was purified by NucleoSpin columns to concentrate DNA and remove salts. Five nanograms of methyl‐enriched DNA underwent library construction using the NEBNext Ultra II DNA Library Prep (New England Biolabs, Ipswich, MA, USA), and the barcoded libraries underwent equimolar pooling and were sent to the Australian Genome Research Facility (AGRF, Melbourne, Australia) for 100 cycles, single end sequencing on HiSeq 2500 (Illumina, San Diego, CA, USA) using version four reagents.
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