CUT&RUN was performed on 0.35×106 TRM-like cells using the ChIC/CUT&RUN Kit (EpiCypher, 14-1048) with 1 μl anti-H3K4me3 (EpiCypher, 13-0041) or 1.5 μl anti-Tcf1 (C46C7, CST) antibodies, following the manufacturer’s protocol, except that 1× eBioscience Perm Buffer (Invitrogen, 00-8333) supplemented with Spermidine and cOmplete™EDTA-free Protease Inhibitor (Roche, 11836170001) was used during the incubation step with pAG-MNase and 0.5× eBioscience Perm Buffer during the wash steps. Libraries were prepared with the NEBNext Ultra II Library Prep Kit (New England Biolabs, E7645) and sequenced as PE150 on Illumina NovaSeq 6000. For data processing, see supplementary materials and methods .
Nebnext ultra 2 library prep kit
Manufactured by New England Biolabs
Sourced in United States
The NEBNext Ultra II Library Prep Kit is a DNA library preparation kit designed for next-generation sequencing. It provides a streamlined workflow for generating high-quality DNA libraries from a variety of input materials, including genomic DNA and PCR amplicons. The kit includes all the necessary reagents and enzymes to perform the library preparation process, including end-repair, dA-tailing, adapter ligation, and PCR amplification.
Automatically generated - may contain errors
Lab products found in correlation
Novaseq 6000, by Illumina (4 mentions)
Hiseq 2500, by Illumina (2 mentions)
Fastselect, by Qiagen (2 mentions)
Zymo pathogen magbead kit, by Zymo Research (2 mentions)
Rnaeasy micro kit, by Qiagen (2 mentions)
Hiseq 2500 system, by Illumina (2 mentions)
Agilent bioanalyser, by Agilent Technologies (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Allprep dna rna kit, by Qiagen (2 mentions)
Dna rna shield, by Zymo Research (2 mentions)
Nextseq 550, by Illumina (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Chic cut run kit, by EpiCypher (1 mentions)
Complete edta free protease inhibitor, by Roche (1 mentions)
Kapa hifi uracil, by Roche (1 mentions)
Agilent 4200 tapestation, by Agilent Technologies (1 mentions)
Zymo gold kit, by Zymo Research (1 mentions)
Qiaamp rna blood mini kit, by Qiagen (1 mentions)
Fragmentase, by New England Biolabs (1 mentions)
28 protocols using nebnext ultra 2 library prep kit
1
CUT&RUN Analysis of H3K4me3 and Tcf1 in TRM-like Cells
2
Targeted Bisulfite Sequencing for Cow Methylomes
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We applied targeted bisulfite sequencing (TBS-seq) to characterize the methylomes of 96 DNA cow samples. The protocol is described in detail in [31 (link)]. Briefly, 500 ng of genomic DNA were used for TBS-seq library preparation. Fragmented DNA was subject to end repair, dA-tailing and adapter ligation using the NEBNext Ultra II Library prep kit (NEB) and custom pre-methylated dual unique index adapters (IDT). Pools of 16 purified libraries were hybridized to 3572 biotinylated probes specific for conserved sequences in mammals (IDT). The sequence of the probes used in this study can be found in Supplementary Table S1.
The Hybridization was carried out using the xGen hybridization capture kit (IDT) according to the manufacturer’s instructions. Captured DNA was bisulfite treated with the Zymo Gold kit (Zymo Research) prior to PCR amplification using KAPA HiFi Uracil+(Roche). The following conditions were used for the PCR amplification: 2 min at 98°C; 14 cycles of (98°C for 20 sec; 60°C for 30 sec; 72°C for 30 sec); 72°C for 5 minutes; hold at 4°C. Library QC was performed using the High-Sensitivity D1000 Assay on a 4200 Agilent TapeStation. Pools of 96 libraries were sequenced on a NovaSeq6000 (Sp lane) as paired-end 150 bases.
The Hybridization was carried out using the xGen hybridization capture kit (IDT) according to the manufacturer’s instructions. Captured DNA was bisulfite treated with the Zymo Gold kit (Zymo Research) prior to PCR amplification using KAPA HiFi Uracil+(Roche). The following conditions were used for the PCR amplification: 2 min at 98°C; 14 cycles of (98°C for 20 sec; 60°C for 30 sec; 72°C for 30 sec); 72°C for 5 minutes; hold at 4°C. Library QC was performed using the High-Sensitivity D1000 Assay on a 4200 Agilent TapeStation. Pools of 96 libraries were sequenced on a NovaSeq6000 (Sp lane) as paired-end 150 bases.
3
Transcriptome Analysis of COVID-19 Patient PBMCs
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We collected PBMC samples from 34 male COVID-19 patients, including 15 non-ICU patients and 19 ICU patients, and 11 healthy males for transcriptome analysis. Transcriptome sequencing of the RNA isolated from whole blood samples was carried out as previously described [12] (link). Briefly, RNA was isolated from whole blood samples using the QIAamp RNA Blood Mini kit (Qiagen, Valencia, CA, USA). The RNA was then reverse-transcribed to generate complementary DNA (cDNA) and was used to construct sequencing libraries using the NEB Next Ultra II Library Prep Kit (New England Biolabs, MA, USA). We used the Illumina HiSeq 2500 for sequencing, and generated 2 × 125 base-read paired-end reads according to the manufacturer’s instructions. The reads were further trimmed to remove low-quality bases. Sequencing reads were mapped against human reference genome GRCh38, and per gene read counts were calculated with TopHat2 (Version 2.1.1) and RSEM (Version 1.2.31). The obtained read counts were normalized using trimmed mean normalization, and differentially expressed genes (DEGs) were estimated using the likelihood ratio test. Functional enrichment analysis was performed based on the list of DEGs using Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources (https:// david.ncifcrf.gov).
4
Metagenomic Sequencing of Biological Samples
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Specimens underwent RNA extraction and metagenomic sequencing, as previously described30 (link). Briefly, RNA was extracted from 200 μL of specimen in DNA/RNA shield using bead-based lysis and the Zymo Pathogen Magbead kit (Zymo). We also processed negative control samples (water and HeLa cell RNA) to account for background contamination. All samples were spiked with RNA standards from the External RNA Controls Consortium (ERCC)44 (link). Samples were DNase treated, depleted of cytosolic and mitochondrial rRNA using FastSelect (Qiagen, Germantown, MD), and reverse transcribed to generate cDNA. Sequencing libraries were constructed using the NEBNext Ultra II Library Prep Kit (New England Biolabs, Ipswich, MA). Libraries underwent 146 nucleotide paired-end sequencing on an Illumina Novaseq 6000 instrument.
5
RNA-seq Analysis of Ketamine Effects
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Two days following 5 days of ketamine or saline injections, brains were rapidly removed and the PLc was dissected and frozen in isopentane on dry ice. Total RNA was isolated using the TriZol reagent (Invitrogen) and purified using RNAeasy micro kits from Qiagen. RNA integrity was assessed using the Agilent Bioanalyser and all RNA integrity number values were above 8. Then, the cDNA library was prepared using the NEB Next Ultra II Library Prep kit (New England Biolabs, USA). Sequencing was conducted on an Illumina HiSeq 2500 system with 100 base pair paired-end reads (London, UK). Raw reads were aligned to mm9 genome using Tophat version (2.0.11) [35 (link)]. Gene based counting was performed using the HTSeq counts module. Gene expression analysis was performed using the DESeq2 Bioconductor package. All genes with adjusted p value of 0.05 or less (calculated from the raw p values using the Benjamini and Hochberg algorithm) were considered statistically significant. The RNA seq data are available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE138802 .
6
Transcriptome Profiling of Tracheal Aspirates
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TA was collected within 24 hours of intubation, mixed 1:1 with DNA/RNA Shield (Zymo), and frozen at –80°C. RNA was extracted from 300 μL patient TA using bead-based lysis and the Allprep DNA/RNA kit (Qiagen), which included a DNase treatment step. RNA was reverse transcribed to generate cDNA, and sequencing library preparation was performed using the NEBNext Ultra II Library Prep Kit (New England BioLabs). RNA-Seq libraries underwent 150 bp paired-end sequencing on an Illumina Novaseq 6000 instrument.
7
Transcriptomic Profiling of Blood Exposure
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Transcriptional sequencing of the RNA isolated from whole blood samples was carried out as described elsewhere (Feng et al., 2021a (link)). The 43 patients were divided into two subgroups, low- and high-exposure groups, based on the exposure level of NO2. Briefly, after generating complementary DNA (cDNA), sequencing libraries were constructed using the NEB Next Ultra II Library Prep Kit (New England Biolabs, MA, USA). Sequencing reads were generated using Illumina HiSeq 2500, and further mapped against human reference genome GRCh38. Per gene read counts were calculated using TopHat2 (Version 2.1.1) and RSEM (Version 1.2.31), and normalized using trimmed mean normalization. Differentially expressed genes (DEG) were estimated using the likelihood ratio test. Functional enrichment analysis was performed based on the list of DEG using Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics Resources (https://david.ncifcrf.gov ).
8
DNA Sequencing Library Preparation
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Sample processing and DNA extraction were carried out as described in the Supplementary Methods. Ten to 100 ng of DNA from each sample was sheared with fragmentase (New England Biolabs) and used to construct sequencing libraries with the NEBNext Ultra II Library Prep Kit (New England Biolabs). Adaptor ligated samples underwent amplification with dual unique indexing primers. Libraries were quantified and pooled and underwent paired end 150 base pair sequencing on an Illumina MiSeq or NextSeq 550. Supplementary Table 2 lists the number of reads obtained for each sample.
9
Ketamine-induced Transcriptomic Changes
10
Illumina Metagenomic and Metatranscriptomic Sequencing
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We closely followed all manufacturer recommendations for the Illumina sequencing platform (Illumina Inc., San Diego, USA). Pooled genomic DNA samples were used to sequence libraries constructed using the NEBNext Ultra II Library Prep Kit (NEB, Ipswich, USA). Paired-end metagenomics sequencing was performed on an NextSeq 550 (Illumina) sequencer using the NextSeq High Output Kit v2 sequencing reagent kit. Metatranscriptome sequencing from pooled RNA samples was performed as follows: libraries were first prepared using a Zymo-Seq RiboFree Total RNA Library Kit, which includes a universal rRNA depletion step. Paired-end mRNA sequencing was then performed on a NextSeq 550 (Illumina) sequencer using the NextSeq High Output Kit v2 sequencing reagent kit. Primary data analysis (i.e., base-calling) was performed using “bcl2fastq” software (version 2.17.1.14, Illumina). Characteristic fragment parameters are summarised in Supplimentary Table 2 .
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