ChIP-seq was carried out as described previously62 (link). Approximately 1 ng ChIP DNA was subjected to library construction using NEBNext Ultra DNA library prep kit (New England Biolabs). Adaptor-ligated DNA was PCR-amplified for 12 cycles using NEBNext Q5 Hot Start HiFi PCR master Mix and NEBNext multiplex oligos (New England Biolabs). PCR products were sequenced on Illumina NextSeq 500 platform to obtain 76-bp single-end reads. Reads were aligned to the human genome (hg19) using Bowtie2 (version 2.2.9). Redundant reads were removed by Picard (version 2.7.1). Reads with low mapping quality (MapQ < 10) were also removed. ChIP-seq peaks were defined by HOMER software (version 4.8.3) using the option “-center -style factor -F 1 -P 0.0001 -fdr 0.05” for RNA Pol II and TP53, while the alternative option “-center -style histone -F 2 -P 0.0001 -fdr 0.05” was used for other proteins. Input DNA described above in the ChIP procedure was also subjected to the same bioinformatic analysis and employed for peak calling; Genomic sections carrying relative enrichment scores between ChIP and Input DNA samples above 2-fold, P < 0.0001 and FDR < 0.05 were defined as peaks.
Nebnext q5 hot start hifi pcr master mix
Manufactured by New England Biolabs
Sourced in United States
NEBNext Q5 Hot Start HiFi PCR Master Mix is a high-fidelity, hot-start PCR master mix formulated for accurate and efficient DNA amplification. It contains the Q5 High-Fidelity DNA Polymerase for enhanced specificity and DNA sequencing accuracy.
Automatically generated - may contain errors
Lab products found in correlation
Nebnext multiplex oligos, by New England Biolabs (5 mentions)
Ampure xp bead, by Beckman Coulter (5 mentions)
Nextseq 500 platform, by Illumina (3 mentions)
Tapestation 2200, by Agilent Technologies (2 mentions)
D1000 screentape, by Agilent Technologies (2 mentions)
Nebnext multiplex oligo kit, by New England Biolabs (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Minelute gel extraction kit, by Qiagen (2 mentions)
Herculase 2 fusion dna polymerase, by Agilent Technologies (2 mentions)
Qiaamp dna blood midi kit, by Qiagen (2 mentions)
Truseq stranded total rna library prep kit, by Illumina (2 mentions)
Labchip rna pico sensitivity assay, by PerkinElmer (2 mentions)
Miseq nano v2 run, by Illumina (2 mentions)
2100 bioanalyzer high sensitivity kit, by Agilent Technologies (2 mentions)
Caliper labchip gx dna high sensitivity reagent kit, by PerkinElmer (2 mentions)
2100 bioanalyzer rna 6000 nano assay, by Agilent Technologies (2 mentions)
Quant it picogreen dsdna assay, by Thermo Fisher Scientific (2 mentions)
Nebnext chip seq library prep reagent set, by Illumina (2 mentions)
Kapa library quantification kit, by Roche (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
26 protocols using nebnext q5 hot start hifi pcr master mix
1
ChIP-seq Protocol for Transcription Factors
2
SARS-CoV-2 S Gene Detection Protocol
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LbCas12a, HiScribe T7 High-Yield RNA Synthesis Kit, RNA Cleanup Kit, NEBNext Q5 Hot Start HiFi PCR Master Mix, NEBuffer2.1 were ordered from NEB (Beijing, China). DNaseI, recombination RNase inhibitor (RRI), PrimeSTAR max were ordered from Takara Bio (Beijing, China). T-vectors, Pfu DNA polymerase were ordered from TIANGEN Biotechnology (Beijing, China). One-Step RT-PCR Kit was ordered from Vazyme Biotech (Nanjing, China). Gel Extraction Kit was ordered from Omega Biotech (Shanghai, China). Lateral flow strips were purchased from Magigen Biotech (Guangzhou, China). Oligonucleotides were synthesized by GENEWIZ (Jiangsu, China). S gene DNA targets were synthesized by Generay Biotech (Shanghai, China). Nucleic acid was quantified using Thermo Fisher Nanodrop 1000 Spectrophotom. Fluorescence signals were recorded with Tecan's Spark 20M.
3
Gut Microbiome Profiling by 16S rRNA Sequencing
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Fresh fecal pellets were collected from each mouse, frozen in liquid nitrogen, and stored at −80°C at the end of the feeding study. DNA was extracted as described previously [17 (link), 18 (link)]. Sequencing of the 16S rRNA genes was performed by the Genomics and Cell Characterization Core Facility at the University of Oregon. Briefly, custom-designed PCR primers that contain dual-indexed adapters were used to amplify the V3–V4 (806R/319F) region using NEBNext® Q5® Hot Start HiFi PCR Master Mix (New England Biolabs, Beverly, MA, USA). Samples were purified by two repeated 0.8X-ratio magnetic bead clean ups to remove all traces of primer. The optimal number of cycles were determined by initial testing on a subset of samples. A no-template control and a ZymoBIOMICS D6305 microbial community DNA standard were included (Zymo Research, Irvine, CA USA). Paired-end 300 bp sequencing was performed on an Illumina MiSeq PE300 (v3 mode) with PhiX added in to about 25%. Approximately 50–60K reads per sample were achieved. Identification of gut microbial amplicon sequence variants (ASVs) followed by chimera removal was performed with DADA2 (v1.16) [55 (link)]. Taxonomy assignment was performed using the Ribosomal Database Project’s Training Set 16 and the 10.28 release of the RDP database [56 (link)]. Raw sequencing data was deposited in the NCBI Gene Expression Omnibus.
4
In Situ Hi-C Protocol for Chromatin Conformation Capture
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In situ Hi-C was performed as previously described9 (link) with slight modifications. Cells at 80% confluence in 10 cm dish were fixed in 1% pFA at room temperature for 10 min and subjected to the in situ Hi-C procedure. After proximal ligation, ligation products were enriched by centrifugation at 10,000 rpm at 4 °C for 10 min. The pellet was suspended with proteinase K solution consisting of 10 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 2 mg/ml proteinase K (Thermo Fisher Scientific) and incubated at 37 °C for 1 h, followed by overnight incubation at 68 °C with 0.5 M NaCl. DNA was purified by phenol/chloroform extraction and sonicated at 4 °C using Bioruptor (Diagenode) for 15 min by repeating the cycle of 30-sec ON and 1-min OFF. Biotin-labeled DNA was purified using Dynabeads (MyOne Streptavidin T1, Thermo Fisher Scientific). Sequencing adapters were ligated using NEBNext Ultra Ligation module (New England Biolabs, Inc) on Dynabeads, and DNA was PCR-amplified for 8–14 cycles using NEBNext Q5 Hot Start HiFi PCR master Mix and NEBNext multiplex oligos (New England Biolabs, Inc). PCR products were sequenced on Illumina NextSeq 500 platform to obtain 76-bp paired-end reads.
5
Optimized HiFi PCR Library Preparation
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NEBNext Q5 Hot Start HiFi PCR Master Mix (NEB, M0543L) and Nextera primers (Buenrostro et al., 2015 ) were used for library preparation. 55 μL of PCR master mix and 5 μL of primer mix were used for 50 μL of samples. Every sample had 25 μM of final concentration by mixing the universal primer Ad1 and the unique index primer 2.X (ex. 2.1, 2.2, 2.3, etc.). The following condition was used for amplification: 65°C, 5 minutes; 98°C, 30 s; 98°C, 10 s, 65°C 30 s, 12 cycles; 4°C indefinitely. 1.5X AMPure XP beads were used for sample purification. The sample concentration was checked using PicoGreen and median fragment size was checked using the Agilent D1000 screentape on the Agilent Technologies 2200 TapeStation. The library was sent to New York Genome Center or MSKCC Integrated Genomics Operation (IGO) center for PE50 sequencing using a HiSeq 2500.
6
Analyzing Lymphocyte Antigen Receptor Rearrangements
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Genomic DNA was purified from sorted cell populations. PCR assays for DH to JH, VH to DJH, or Vκ to Jκ rearrangements were adapted from ref. 51 (link) to work with 1 ng of total material. Briefly, PCR was set according to the NEBNext Q5 Hot Start HiFi PCR Master Mix (New England Biolabs) manufacturing protocol. PCR products were analyzed on an agarose gel, blotted to Amersham Hybond-N+ membrane (GE Healthcare), and followed by southern blotting.
Southern blot was carried out as previously described (52 (link)). Briefly, blots were prehybridized at 42 °C for 1 h and then, hybridized overnight using a P32 end-labeled oligonucleotide probe. Blots were washed with 2× saline-sodium citrate + 0.05% sodium dodecyl sulfate (SDS) for 20 min four times, and radioactive signals were detected by a GE Healthcare Amersham Typhoon imager. The nonrearranging RAG1 locus served as a loading control. Lane 9 is an amplification reaction with no genomic DNA. Samples in lanes 10 and 11 were used as controls: whole wild-type C57BL/6N bone marrow extract and Abelson-transformed RAG2−/− pro-B lymphocytes.
Southern blot was carried out as previously described (52 (link)). Briefly, blots were prehybridized at 42 °C for 1 h and then, hybridized overnight using a P32 end-labeled oligonucleotide probe. Blots were washed with 2× saline-sodium citrate + 0.05% sodium dodecyl sulfate (SDS) for 20 min four times, and radioactive signals were detected by a GE Healthcare Amersham Typhoon imager. The nonrearranging RAG1 locus served as a loading control. Lane 9 is an amplification reaction with no genomic DNA. Samples in lanes 10 and 11 were used as controls: whole wild-type C57BL/6N bone marrow extract and Abelson-transformed RAG2−/− pro-B lymphocytes.
7
Illumina Library Preparation Protocol
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A total of 50 μL from all six DNA extracts (Lo5h, Lo2, Lo3, Q6h, Q1, Q2) and four reagent controls (RB1 to RB4) were converted into double stranded DNA Illumina libraries using the NEBNext® UltraTM II DNA Library Prep Kit for Illumina® (NEB, Ipswich, MA, USA) and looped adapters from the NEBNext® Multiplex Oligos for Illumina® kit (NEB). Illumina libraries were prepared according to the manufacturer’s instructions except for ligation which used highly diluted adapters and was performed overnight at 7 °C. Following ligation, the ligated looped adapters were converted into Y-shaped adapters with the USER® Enzyme kit (NEB). The libraries were then purified with 1.8× AMPure XP beads (Beckman Coulter, Sykesville, MD, USA). All the libraries were dual indexed with primers from the NEBNext® Multiplex Oligos for Illumina® kit and subsequently amplified for 25 cycles with the NEBNext® Q5® Hot Start HiFi PCR Master Mix (NEB). Purification of the amplified libraries was performed using 1.5× AMPure XP beads. The libraries were then shotgun sequenced on an MiSeq Forensic Genomics System (FGxTM) instrument (Illumina Inc., San Diego, CA, USA) with a v2 cartridge and 2 × 151 cycles and/or on a NextSeq 500/550 (Illumina) with a High Output kit v2.5 and 2 × 75 cycles in order to obtain additional data.
8
Tau cDNA Library Construction
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Tau cDNA (NM_016,834.4) was amplified from Tau-LexA bait vector (oligonucleotides 6690 and 6972 in Figure S18 ); 5 μg of the PCR product was subjected to Fragmentase treatment (New England Biolab, NEB) until a smear of fragments was detected around 400 to 500 pb by agarose gel electrophoresis. The DNA fragments were purified by phenol/chloroform extraction and ethanol precipitation. The DNA fragments were next subjected to end repair (NEB) and dA-tailing adaptation, using Blunt/TA ligase master mix with NEBNext Adaptor hairpin loop (NEB), followed by AMPure XP bead (Beckman Coulter) purification. After USER enzyme digestion (NEB), DNA fragments were amplified (oligonucleotides 10829 and 10830 in Figure S18 ) with 15 cycles of PCR using NEBNext Q5 Hot Start HiFi PCR Master Mix (NEB), which allowed addition Gap Repair recombination sequences for the cloning in Gal4-AD prey plasmid pP7. The library comprised 50,000 independent clones.
9
16S rRNA sequencing for microbiome analysis
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For marker gene‐based microbiome analysis, the V4 regions (515F‐806R) of the bacterial 16S rRNA genes were amplified using the NEBNext Q5 Hot Start Hifi PCR Master Mix (New England Biolabs, Frankfurt am Main, Germany) using protocols established in the Earth microbiome project (https://earthmicrobiome.org/protocols‐and‐standards/16s/ ). Amplified fragments were purified with AMPure XP Beads (Beckmann Coulter GmbH, Krefeld, Germany), pooled in equimolar ratios and analyzed by 2 × 151 bp paired‐end sequencing on an Illumina MiSeq device (Illumina Inc., San Diego, USA). Raw fastq files were then imported and analyzed in QIIME2 v2021.8 (Bolyen et al, 2019 (link)) with Dada2 (Callahan et al, 2016 (link)) as the method for quality control, dereplication, and sub‐operating taxonomic unit/amplicon sequence variant (sOTU/ASV) table generation. The SILVA (Quast et al, 2013 (link)) small subunit database release 138 was used at a 99% similarity cutoff for taxonomic classification.
10
Sequencing and Phylogenetic Analysis of PPMV-1 and SARS-CoV-2
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PPMV-1 genes were amplified by RT-PCR and library preparation using a Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA) for sequencing on the Illumina MiniSeq platform. Raw FastQ data for PPMV-1 were assembled using a CLC Genomics Workbench, version 21.0, and the BLAST sequence was searched against the NCBI database. MEGA 6.0 was used for sequence alignment and construction of a phylogenetic tree based on the F gene with PPMV-1 reference sequences downloaded from the NCBI. In the same way, SARS-CoV-2 RNA was amplified by RT-PCR using upstream and downstream primer combinations covering the entire genome and NEBNext Q5 hot start HiFi PCR master mix (New England Biolabs, Hitchin, UK). After library preparation, the genome was sequenced on the Illumina MiniSeq platform. Raw FastQ data for SARS-CoV-2 were assembled using the CLC Genomics Workbench, version 21.0, and the censuses sequence was uploaded to https://pangolin.cog-uk.io/ for type identification.
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