Ultra 2 q5 master mix

Manufactured by New England Biolabs

The Ultra II Q5 Master Mix is a high-fidelity, ready-to-use PCR reagent formulation designed for reliable and efficient DNA amplification. It contains the Q5 High-Fidelity DNA Polymerase, optimized buffer, and dNTPs.

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Lab products found in correlation

Csp6i, by Thermo Fisher Scientific (1 mentions) Nlaiii, by New England Biolabs (1 mentions) Ampure xp, by Beckman Coulter (1 mentions) Tianamp genomic dna kit, by Tiangen Biotech (1 mentions) Thermocycler, by Bio-Rad (1 mentions) Hiseq x ten, by Illumina (1 mentions) Dna clean concentrator kit, by Zymo Research (1 mentions) Novaseq, by Illumina (1 mentions) Nextseq, by Illumina (1 mentions) Monarch dna clean up kits, by New England Biolabs (1 mentions) Nextera xt dna library preparation kit, by Illumina (1 mentions) Bioanalyzer, by Agilent Technologies (1 mentions)

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Q5 master mix (39 citations) Ultra II (3 citations)

6 protocols using ultra 2 q5 master mix

Chromosome Conformation Capture Sequencing

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4C sample preparation was performed as previously described by Splinter et al. using NlaIII (NEB) and Csp6I (Thermo)^{34 (link)}. Viewpoint primers were selected from a previously reported database^{35 (link)}.
Primers sequences were concatenated with Illumina compatible adapter sequences and unique barcodes and synthesized as Ultramer oligos (IDT). Final library amplification was set up on ice using 3.2 µg of purified 4C sample DNA, 2.5 µM primers, and Ultra II Q5 Master Mix (NEB) split into 16 × 50 µl PCR reactions with extension at 65 °C. Amplification products were purified using 1.5× Ampure XP (Beckman) bead purification. Size distributions were established using a Bioanalyzer DNA 12000 Assay (Agilent).
Paired-end sequencing reads were generated as above and analyzed with the 4C-ker pipeline as previously described to identify genomic loci with differential chromosome contacts between conditions^{36 (link)}.

Tarjan D.R., Flavahan W.A, & Bernstein B.E. (2019). Epigenome editing strategies for the functional annotation of CTCF insulators. Nature Communications, 10, 4258.

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Genomic DNA Extraction and gRNA NGS Library Preparation

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Genomic DNA extraction was performed using TIANamp Genomic DNA kit (TIANGEN) and finally resuspended in 50 μl nuclease free water. To prepare the gRNA NGS library for the SR sample, all gDNA were amplified on thermocycling with parameters of 98°C for 30 s, 20–22 cycles of (98°C for 10 s, 64°C for 30 s, 72°C for 20 s), 72°C for 2 mins. To prepare the gRNA NGS library for the TIL sample, two-step amplification was applied. In the first step, PCR reaction (400–800 ng DNA input per reaction, 2–4 reactions per sample) was performed using Ultra II Q5 Master Mix (NEB) with thermocycling parameters as 98°C for 30 s, 28–30 cycles of (98°C for 10 s, 60°C for 30 s, 72°C for 20 s), 72°C for 2 mins. And the PCR condition and primers of the second step follows the condition of the SR library preparation, but with 8–10 cycles. All primers used in NGS library preparation were listed in Supplementary Table S1. Both G12 and G23 NGS libraries were prepared for the TIL sample as stated in the GGA4 NGS library preparation methods part.

Lu Z., Ni K., Wang Y., Zhou Y., Li Y., Yan J., Song Q., Liu M., Xu Y., Yu Z., Guo T, & Ma L. (2022). An in-library ligation strategy and its application in CRISPR/Cas9 screening of high-order gRNA combinations. Nucleic Acids Research, 50(11), 6575-6586.

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RNase H-based cDNA Library Preparation

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One microliter of RNase H buffer and 1 μl of RNase H were added into the resuspended reverse transcription product and placed in a thermocycler (Bio-Rad) at 37 °C for 30 min. Beads were washed and resuspended in 50 μl of water. cDNA was eluted by boiling the beads at 95 °C for 10 min, purified using a DNA Clean & Concentrator kit (Zymo Research) to remove short adapters and eluted into 10 μl of water. Two microliters of 10× T4 RNA ligase buffer, 2 μl of 10 mM ATP, 10 μl of 50% PEG8000, 1 μl of cDNA_3′adapter (50 μM; Supplementary Table 1) and 1 μl of T4 RNA ligase 1 were added into the eluted cDNA, and the ligation was performed at 25 °C overnight. The reaction was purified using a DNA Clean & Concentrator kit (Zymo Research) and eluted with 21 μl of water. One microliter of supernatant was used for quantitative real-time PCR (qPCR) testing, and the remaining 15 μl was used for library construction. NEBNext Ultra II Q5 Master Mix and NEBNext adaptors were used for library amplification. Amplified libraries were purified using 0.8× Ampure beads. The purified libraries were sent for next-generation deep sequencing. The libraries were sequenced on an Illumina HiSeq X Ten with paired-end 2 × 150 bp read length.

Hu L., Liu S., Peng Y., Ge R., Su R., Senevirathne C., Harada B.T., Dai Q., Wei J., Zhang L., Hao Z., Luo L., Wang H., Wang Y., Luo M., Chen M., Chen J, & He C. (2022). m6A RNA modifications are measured at single-base resolution across the mammalian transcriptome. Nature biotechnology, 40(8), 1210-1219.

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Illumina Library Preparation and Sequencing

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Libraries were indexed with NEBNext Multiple Oligos for Illumina (NEB #E6440) and amplified using NEBNext Ultra II Q5 Master Mix for 5 cycles. The quality and quantity of libraries were checked with Agilent TapeStation Systems with High Sensitivity D1000 reagent and screen tape (Agilent #5067–5585 and Agilent #5067–5584). The libraries were pooled for sequencing on Illumina Nextseq instrument or Illumina Novaseq instrument with paired-end reads of 75 bp or 100 bp. For each sample, we achieved 10–20 million reads.
Reads were downloaded as FASTQ files and trimmed with Trim Galore v0.6.4 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The option—paired were used when processing paired end reads. Trimmed reads were aligned to the reference genome with BOWTIE2 v2.3.5.1 (26), together with SAMTOOLS V1.9 [32 (link)] to sort and index bam files for later analysis.

Yang W., Fomenkov A., Heiter D., Xu S.Y, & Ettwiller L. (2022). High-throughput sequencing of EcoWI restriction fragments maps the genome-wide landscape of phosphorothioate modification at base resolution. PLoS Genetics, 18(9), e1010389.

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SARS-CoV-2 Genome Amplification and Sequencing

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Amplicons tiling the SARS-CoV-2 genome were generated using NEBNext UltraII Q5 MasterMix (Cat. No. M0544L), gene-specific forward and reverse PCR primers, and 5μL of purified cDNA. Gene-specific primers used for PCR are listed in Table S3. Touchdown cycling PCR conditions were used to enhance PCR specificity (68–58°C annealing temperature gradient). PCR reaction products were purified with Monarch DNA Clean-up Kits (NEB) with a binding buffer:sample ratio of 2:1 to remove products smaller than 2kb. PCR products were visualized on 0.8% agarose gels to confirm production of correctly sized amplicons. Amplicons were diluted to 0.2ng/uL and then pooled into two odd and two even amplicon pools for downstream library preparation. Sequencing libraries were generated using a NexteraXT DNA Library Preparation Kit (Illumina) according to manufacturer’s protocol, but with 1/5^th the recommended volume. Libraries were quantified using a Qubit (Life Technologies) and a BioAnalyzer (Agilent). Amplicon pools were recombined and sequenced on a NextSeq 500/550 platform using a 150 cycle mid-output kit.

Huston N.C., Wan H., de Cesaris Araujo Tavares R., Wilen C, & Pyle A.M. (2020). Comprehensive in-vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms. bioRxiv.

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UMI-Tagged VH and VL Amplification

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Post ligation, UMI and dual UMI-tagged VH and VL were amplified using 25 μl NEBNext Ultra II Q5 Master Mix, 5 μl of the ligation product and the following primers in a final volume of 50 μl: for Mix9, 5 μl of 10 μM primers FabK_VH_NGSCS2_Fw, FabKL_VH_NGSCS1_Rv each for VH-UMI and primers FabK_VL_NGSCS1_Fw, FabK_VL_NGSCS2_Rv each for VL-UMI. For Mix14 and YSD Fab libraries, 5 μl of 10 μM primers FabNGS_VH0N_CS2_Fw, KVH0N_CS1_Rv each for VH dual-UMI and primers FabNGS_VL0N_CS1_Fw, FabNGS_VL0N_CS2_Rv each for VL dual-UMI. The cycling conditions included 30 s at 98°C for initial denaturation, followed by 10 s denaturation at 98°C, annealing at 70°C for 20 s and extension at 72°C for 25 s for 18–20 cycles, finalized by extension at 72°C for 2 min. The amplified UMI and dual UMI-tagged VH and VL fragments were purified on a Nucleospin column according to the manufacturer’s instructions.

Levin I., Štrajbl M., Fastman Y., Baran D., Twito S., Mioduser J., Keren A., Fischman S., Zhenin M., Nimrod G., Levitin N., Mayor M.B., Gadrich M, & Ofran Y. (2023). Accurate profiling of full-length Fv in highly homologous antibody libraries using UMI tagged short reads. Nucleic Acids Research, 51(11), e61.

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