Nebnext ultra 2 q5 master mix

Manufactured by New England Biolabs

Sourced in United States

The NEBNext Ultra II Q5 Master Mix is a highly-concentrated, ready-to-use mixture of reagents designed for high-fidelity PCR amplification. It contains the Q5 High-Fidelity DNA Polymerase, dNTPs, and necessary buffers.

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Lab products found in correlation

Ampure xp bead, by Beckman Coulter (8 mentions) Dna clean concentrator 5 kit, by Zymo Research (5 mentions) Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions) Purelink quick gel extraction kit, by Thermo Fisher Scientific (2 mentions) Cutsmart buffer, by New England Biolabs (2 mentions) Nebuilder hifi dna assembly master mix, by New England Biolabs (2 mentions) 2100 bioanalyzer instrument, by Thermo Fisher Scientific (2 mentions) Qubit ssdna high sensitivity assay kit, by Thermo Fisher Scientific (2 mentions) Agencourt ampure xp spri beads, by Beckman Coulter (2 mentions) Miniseq, by Illumina (2 mentions) Onetaq dna polymerase, by New England Biolabs (2 mentions) Nucleospin tissue kit, by Macherey-Nagel (2 mentions) Dneasy blood tissue kit, by Qiagen (2 mentions) Bioanalyzer, by Agilent Technologies (2 mentions) Tapestation 2200, by Agilent Technologies (2 mentions) Bluepippin gel, by Sage Science (2 mentions) M220 focused ultrasonicator, by Covaris (2 mentions) Q5 hot start high fidelity 2x master mix, by New England Biolabs (2 mentions) Tn5 transposase, by Illumina (2 mentions) Dynabeads myone streptavidin c1, by Illumina (2 mentions)

57 protocols using nebnext ultra 2 q5 master mix

5′ RNA Sequencing and Analysis

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The 5′ RNA sequencing was performed by ImmunoGeneTeqs, Inc. (Chiba, Japan). PolyA⁺ RNA was isolated using Dynabeads M-270 Streptavidin (Thermo Fisher Scientific) and biotin-3′ WTA-EcoP-dT25, according to the previous analysis (GSE110711) with some modifications. After reverse transcription and template switching, the cDNA was amplified and subjected to fragmentation/end-repair/polyA-tailing/ligation using NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs). Barcoded libraries (~300 bp) were obtained by PCR using NEBNext Ultra IIQ5 Master Mix (New England Biolabs), and sequenced on the Illumina Novaseq 6000 S4 flowcell (Illumina). After adaptor trimming of sequencing data in single-end fastq files using cutadapt 2.10, the reads were mapped to reference RNA (mRNA and ncRNA of GRCm38 release 101) with bowtie 2-2.3.4.2. The reads in each gene were counted using awk, sort and uniq -c commands. The count data was summarized, and the expression table was full-outer joined by gene symbols using Microsoft R open-3.5.3 and dplyr-1.0.0 package.

Yamazaki S., Inohara N., Ohmuraya M., Tsuneoka Y., Yagita H., Katagiri T., Nishina T., Mikami T., Funato H., Araki K, & Nakano H. (2022). IκBζ controls IL-17-triggered gene expression program in intestinal epithelial cells that restricts colonization of SFB and prevents Th17-associated pathologies. Mucosal Immunology, 15(6), 1321-1337.

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Illumina RNA-Seq Library Preparation

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The double‐stranded cDNA pool was amplified by PCR using NEBNext Ultra II Q5 Master Mix (NEB, #M0544L) with 0.5 µm Illumina RP1 primer and Illumina RPIx primer (where x is a number indicating the Illumina index). The thermocycling protocol began with 98 °C for 30 s followed by 12 cycles of 98 °C for 15 s, 62 °C for 15 s, and 72 °C for 60 s, and then incubated at 72 °C for 7 min. Three replicates of Illumina TruSeq library (Illumina, #20020594) for am or pm respectively were constructed starting with the same total RNA and ERCC Ex‐Fold Spike‐Ins with SiPAS libraries following the manufacturer’s instructions. The libraries were sequenced using the Illumina NovoSeq platform with PE150 sequencing format.

Wang J., Xu J., Yang X., Xu S., Zhang M, & Lu F. (2021). Boosting the power of transcriptomics by developing an efficient gene expression profiling approach. Plant Biotechnology Journal, 20(1), 201-210.

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Two-Stage PCR for ITS Amplicon Sequencing

Cited 1 time

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NGS libraries were prepared using two-stage PCR [61 (link),62 (link)] using ITS1 and ITS2 primers fused with Illumina Nextera adapters to simplify library preparation, which indicated the following: the primer for ITS1 (forward 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGGAAGGAGAAGTCGTAACAAGG-3′; reverse 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGAGATATCCGTTGCCGAGAGT-3′) and the primer for ITS2 (forward 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGATCGAGTYTTTGAACGCAAGTTG-3′; reverse 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCTCCGCTTATTGATATGCT-3′). The first PCR of pollen DNA samples conducted to obtain ITS1 and ITS2 amplicons was performed using Encyclo Plus PCR Kit (Evrogen, Moscow, Russia). The second PCR for library indexing was performed with the NEBNext Ultra II Q5 Master Mix (NEB, Ipswich, MA, USA) kit and Nextera XT Index Kit v2 Set A (Illumina, San Diego, CA, USA). After each amplification, DNA was purified using AMPure beads with a 1.1× bead ratio. Purified amplicons were quantified via fluorimetry using Qubit dsDNA HS Assay Kit and a Qubit 3.0 instrument.
High-throughput sequencing was performed on the Illumina MiSeq platform with MiSeq Reagent Kit v3, 2 × 300 nt paired-end (Illumina, San Diego, CA, USA).

Krinitsina A.A., Omelchenko D.O., Kasianov A.S., Karaseva V.S., Selezneva Y.M., Chesnokova O.V., Shirobokov V.A., Polevova S.V, & Severova E.E. (2023). Aerobiological Monitoring and Metabarcoding of Grass Pollen. Plants, 12(12), 2351.

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Barcoded CRISPR and GWAS Arrays

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The CMS array oligos were synthesized by CustomArray and the GWAS array oligos were synthesized by Twist Biosciences. Post-synthesis, for the CMS array, 6 bp random hexamer barcodes and additional adapter sequences were added by performing 20 PCR reactions, each 50 μL in volume, containing 5.7 ng of oligo, 25 μL of Q5 NEBNext MasterMix (NEB, M0541S), 1 unit Q5 HotStart polymerase (NEB, M0493S), 0.5 μM oligo_BAR_Bmt_F and oligo_pmir_R_min primers, and 20 μg BSA (NEB, B9000) (Supplementary Table 5). PCR cycling conditions used are as follows: 98°C for 30 seconds, 22 cycles (98°C for 30 sec, 60°C for 30 sec, and 72°C for 1 min), 72°C for 2 min. For the GWAS array oligos, PCR was performed using the same primers and cycling conditions with the following exceptions: 1) 24 50 μL PCR reactions instead of 20, 2) 1 ng of oligo in each reaction instead of 5.7 ng, 3) use of the NEBNext Ultra II Q5 Master Mix (NEB, M0544L) instead of the Q5 HotStart polymerase, 4) and 12 cycles for amplification instead of 22 cycles. PCR reactions from both arrays were purified by performing a 2.5X SPRI purification using Agencourt AMPure XP SPRI (Beckman Coulter, A63881) beads according to manufacturer instructions.

Griesemer D., Xue J.R., Reilly S.K., Ulirsch J.C., Kukreja K., Davis J.R., Kanai M., Yang D.K., Butts J.C., Guney M.H., Luban J., Montgomery S.B., Finucane H.K., Novina C.D., Tewhey R, & Sabeti P.C. (2021). Genome-wide functional screen of 3’UTR variants uncovers causal variants for human disease and evolution. Cell, 184(20), 5247-5260.e19.

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Bacterial DNA Extraction and 16S Sequencing

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Total DNA was extracted using a Hipure Bacterial DNA kit (Magen, D3146; China), according to manufacturer's instructions. This was inspected using an ultramicrospectrophotometer (K5500, Kai'ao, China), and by agarose gel electrophoresis (Qubit® dsDNA HS Assay Kit, Life Technologies, USA; Qubit 2.0, Life Technologies, USA). Universal primers for 16S variable regions V3-V4 were used for the polymerase chain reaction (PCR) amplification. The specific primers inside and outside were amplified by multiplex PCR assay (NEBnext-Ultra-II-Q5-Master-Mix, NEB, USA) and inspected by employing the Agilent 2200 Tapestation system (Aglient Technologies, USA). These samples were prepared according to the Miseq User Guide (Miseq Reagent Kit V3; 600 Cycle PE, Illumina, USA), which mainly included four steps: library preparation, cluster generation, sequencing, and data analysis. Subsequently, the sequencing of the paired-end (2X300) was conducted according to the Miseq User Guide.

Lin W.X., Du X., Yang L.L., Chen S.Y., Qiu W.Y., Wu H.W., Zhao G., Feng Y.H., Yu Q.Y., Tian H., Luo S.P, & Gao J. (2019). Differences in the Composition of Vaginal Microbiota between Women Exhibiting Spleen-Deficiency Syndrome and Women with Damp-Heat Syndrome, Two of the Most Common Syndromes of Vaginitis in Traditional Chinese Medicine. Evidence-based Complementary and Alternative Medicine : eCAM, 2019, 5456379.

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High-throughput Enhancer Screening Assay

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One-hundred nanograms of a 350-nt DNA oligonucleotide pool containing 500 insert/barcode combinations (IDT) (Supplementary Table S2) were PCR-amplified in 50 μl volume [25 μl NEBNext Ultra II Q5 Master Mix (NEB), 0.25 μl 100 μM pMPRA1-LH (5′-GGTAACCGGTCCAGCTCA-3′), 0.25 μl 100 μM pMPRA1-RH (5′-CGTGTGCTCTTCCGATCT-3′)] with the following conditions: 30 s at 98°C, followed by 15x cycles of [10 s at 98°C, 20 s at 65°C and 15 s at 72°C], and finally 2 min at 72°C. The PCR product (350 bp) was run on a 2% TBE/agarose gel, then gel-extracted and cleaned up using PureLink Quick Gel Extraction Kit (Invitrogen; K210012). Four-hundred nanograms pTSS-MPRA-Empty plasmid were digested with BsaI in 20 μl [1 μl BsaI, 2 μl CutSmart Buffer (NEB)] at 37°C for 1 h. Cut plasmid was gel-extracted and cleaned up using PureLink Quick Gel Extraction Kit (Invitrogen; K210012). Amplified library was Gibson-assembled into cut plasmid using NEBuilder HiFi DNA Assembly Master Mix (NEB) with a 5-fold molar excess of the library at 50°C for 1 hour in 4 μl total volume (higher complexity library assembly should be carried out in higher volumes).

Guzman C., Duttke S., Zhu Y., De Arruda Saldanha C., Downes N.L., Benner C, & Heinz S. (2023). Combining TSS-MPRA and sensitive TSS profile dissimilarity scoring to study the sequence determinants of transcription initiation. Nucleic Acids Research, 51(15), e80.

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Multiplex Barcoding for Single-Cell Sequencing

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For both single-cell DNA and RNA experiments, the last round of BAG barcode was added in the same way. Many strategies can be used to perform split-and-pool (Cao et al. 2017 (link); Vitak et al. 2017 (link); Rosenberg et al. 2018 (link); Cao et al. 2019 (link)), and additional rounds of BAG split-barcodes can be added based on the common sequence from the previous round (Supplemental Method S2). Here we used PCR to add 96 different BAG barcodes in the last split. In each well, there is a universal PCR primer and a well-specific primer containing different barcodes. BAGs from the last split-and-pool were evenly distributed into 96 wells. DNA BAGs were amplified using NEBNext ultra II Q5 master mix (NEB M0544). RNA BAGs were amplified using KAPA HiFi HotStart ReadyMix (Roche KK2602). The PCR product was pooled together and purified using AMPure XP magnetic beads (Bechman Coulter A63881).

Li S., Kendall J., Park S., Wang Z., Alexander J., Moffitt A., Ranade N., Danyko C., Gegenhuber B., Fischer S., Robinson B.D., Lepor H., Tollkuhn J., Gillis J., Brouzes E., Krasnitz A., Levy D, & Wigler M. (2020). Copolymerization of single-cell nucleic acids into balls of acrylamide gel. Genome Research, 30(1), 49-61.

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Gibson Assembly of Plasmid Library

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Each sequence was PCR-amplified from K562 genomic DNA in 50 μl volume [25 μl NEBNext Ultra II Q5 Master Mix (NEB), 0.25 μl 100 μM Fwd Primer (see Supplementary Table S3), 0.25 μl 100 μM Rev Primer (see Supplementary Table S3), 40 ng K562 genomic DNA] with the following conditions: 1 minute at 98°C, followed by 12× cycles of [10 s at 98°C, 30 s at 70°C and 30 s at 72°C], and finally 2 min at 72°C. PCR product was isolated with 10% (w/v) PEG 8000/SpeedBeads [2 μl SpeedBeads E3 (Cytiva, cat# 65152105050250) + 48 μl 20% (w/v) PEG 8000], beads were washed 2× with 80% ethanol and air-dried at RT for 12 min, and DNA eluted by resuspending in LoTE [1:4-diluted Tris-EDTA buffer pH 7.5]. Four-hundred nanograms pTSS-MPRA-Empty plasmid were digested with BsaI in 20 μl [1 μl BsaI, 2 μl CutSmart Buffer (NEB)] at 37°C for 1 h. Cut plasmid was gel-extracted and cleaned up using PureLink Quick Gel Extraction Kit (Invitrogen; K210012). Amplified sequences were Gibson-assembled into cut plasmid using NEBuilder HiFi DNA Assembly Master Mix (NEB) with a 2.5-fold molar excess of the library at 50°C for 1 h in 4 μl total volume.

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Amplification and Barcoding of sgRNAs for Cell Population Analysis

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Amplification and bar-coding of sgRNAs was performed as previously described⁴³ with some modifications. Briefly, after gDNA isolation, sgRNAs were amplified and barcoded with TruSeq Single Indexes using a one-step PCR. TruSeq Adaptor Index 12 (CTTGTA) was used for the Foxp3^low population and TrueSeq Adaptor Index 14 (AGTTCC) was used for the Foxp3^high population. Each PCR reaction consisted of 50μL of NEBNext Ultra II Q5 Master Mix (NEB, Cat# M0544), 1μg of gDNA, 2.5μL each of the 10μM forward and reverse primers, and water to 100μL total. The PCR cycling conditions were: 3 minutes at 98°C, followed by 10 seconds at 98°C, 10 seconds at 62°C, 25 seconds at 72°C, for 26 cycles; and a final 2 minute extension at 72°C. After the PCR, the samples were purified using Agencourt AMPure XP SPRI beads (Beckman Coulter, Cat #A63880) per the manufacturer’s protocol, quantified using the Qubit ssDNA high sensitivity assay kit (Thermo Fisher Scientific, Cat #Q32854), and then analyzed on the 2100 Bioanalyzer Instrument. Samples were then sequenced on an Illumina MiniSeq using a custom sequencing primer. Primer sequences are listed in Supplementary Table 3.

Cortez J.T., Montauti E., Shifrut E., Gatchalian J., Zhang Y., Shaked O., Xu Y., Roth T.L., Simeonov D.R., Zhang Y., Chen S., Li Z., Woo J.M., Ho J., Vogel I.A., Prator G.Y., Zhang B., Lee Y., Sun Z., Ifergan I., Van Gool F., Hargreaves D.C., Bluestone J.A., Marson A, & Fang D. (2020). CRISPR Screen in Regulatory T Cells Reveals Modulators of Foxp3. Nature, 582(7812), 416-420.

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Amplicon Sequencing Library Preparation

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NucleoSpin^® Tissue Kit (MACHEREY‐NAGEL) was used to isolate genomic DNA according to the manufacturer's instructions. Barcodes were amplified from genomic DNA with P5.seq‐B‐GLI.v1 and P7.seq‐B‐GLI.v1 primers using OneTaq^® DNA Polymerase (NEB; catalog number M0480). Reactions were purified using NucleoSpin^® Gel and PCR Clean‐up Kit (MACHEREY‐NAGEL). Then, purified amplicons were amplified with primers, Illumina_indX_F and Illumina_indX_R (where X indicates the index sequence), to add Illumina adapters and indexes for sample multiplexing. This round of PCRs was performed using NEBNext^® Ultra™ II Q5^® Master Mix (NEB, catalog number M0544). Samples were purified using AMPure XP beads (Beckman Coulter; catalog number A63880). Next‐generation sequencing library was sequenced with HiSeq 2500 Illumina sequencer using 100‐bp paired‐end protocol (with 10% PhiX DNA spike‐in). To improve cluster calling, we increased sequence diversity by using a 15‐bp random sequence stagger in the P5.seq‐B‐GLI.v1 primer.

Akimov Y., Bulanova D., Timonen S., Wennerberg K, & Aittokallio T. (2020). Improved detection of differentially represented DNA barcodes for high‐throughput clonal phenomics. Molecular Systems Biology, 16(3), e9195.

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