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Kapa hifi 2x readymix

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KAPA HiFi 2x Readymix is a pre-formulated, ready-to-use PCR master mix optimized for high-fidelity DNA amplification. It is designed to provide consistent and reliable performance for a wide range of applications.

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Lab products found in correlation

Lambda exonuclease, by New England Biolabs (2 mentions) Rnase a, by Thermo Fisher Scientific (2 mentions) Smartscribe reverse transcriptase, by Takara Bio (2 mentions) Sybr green, by Thermo Fisher Scientific (2 mentions) Zymopure 2 plasmid midiprep kit, by Zymo Research (2 mentions) Nebuilder hifi dna assembly cloning kit, by New England Biolabs (2 mentions) Monarch pcr dna cleanup kit, by New England Biolabs (2 mentions) Dneasy blood tissue kit, by Qiagen (2 mentions) Monarch plasmid miniprep kit, by New England Biolabs (2 mentions) Nextseq 550 high output kit, by Illumina (2 mentions) Ampure bead, by Beckman Coulter (2 mentions) Tapestation hs d1000 kit, by Agilent Technologies (2 mentions) Dynabeads mrna direct purification kit, by Thermo Fisher Scientific (2 mentions) Maxima h minus reverse transcriptase, by Thermo Fisher Scientific (2 mentions) Dnase 1, by Thermo Fisher Scientific (2 mentions) Nextera xt dna library prep kit, by Illumina (2 mentions) Direct zol rna microprep kit, by Zymo Research (2 mentions) Agencourt ampure xp spri beads, by Beckman Coulter (1 mentions) Expand long template pcr system, by Merck Group (1 mentions)

9 protocols using kapa hifi 2x readymix

1

Single-Cell RNA Sequencing of B Cells

Cited 2 times
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All study protocols were approved by the Institutional Review Board at University of California, Santa Cruz (UCSC). A blood sample of a fully consented healthy adult was collected at UCSC. The sample was deidentified upon collection. B cells were isolated by FACS (13 (link)). RNA and cell lysates were amplified using the Tn5Prime (13 (link)) method, which represents a modification of the Smart-seq2 (18 (link), 19 (link)) method. Briefly, 100 pg of SIRV E2 (Lexogen) RNA or single B cell lysates were reverse-transcribed (RT) using Smartscribe Reverse Transcriptase (Clontech) in a 10-μL reaction including an oligo(dT) primer and a Nextera A TSO containing a 7-nt sample index (SI Appendix, Table S3). RT was performed for 60 min at 42 °C. The resulting cDNA was treated with 1 μL of 1:10 dilutions of RNase A (Thermo) and Lambda Exonuclease New England Biolabs (NEB) for 30 min at 37 °C. The treated cDNA was then amplified using KAPA HiFi Readymix 2x (KAPA) and incubated at 95 °C for 3 min, followed by 15 cycles for SIRV RNA or 27 cycles (single B cells) of 98 °C for 20 s, 67 °C for 15 s, and 72 °C for 4 min, with a final extension at 72 °C for 5 min. cDNA amplification requires both the ISPCR primer and a Nextera A Index primer, which contains another 8-nt sample index.
Volden R., Palmer T., Byrne A., Cole C., Schmitz R.J., Green R.E, & Vollmers C. (2018). Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA. Proceedings of the National Academy of Sciences of the United States of America, 115(39), 9726-9731.
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2

Comprehensive Full-Length cDNA Generation

Cited 1 time
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RNA was reverse transcribed (RT) using SMARTScribe Reverse Transcriptase (Clonetech). We generated full-length cDNA using a modified Smart-seq2 approach (Cole et al., 2018 (link)). During the RT reaction, a template-switch oligo and an oligodT primer were used to select for polyA+ RNA (Table S2). The RT reaction was performed in 10 μl reactions with an input of 70 ng of RNA and took place at 42°C for 1 h. After cDNA synthesis, 1 μl of 1:10 dilutions of RNAse A (Thermo Fisher) and Lambda Exonuclease (NEB) were added and incubated at 37°C for 30 min. Following the incubation, an amplification step was performed in 25-μl final volumes using KAPA HiFi ReadyMix 2X (Kapa Biosystems) containing 1 μl of the ISPCR primer (10 μM) primer. Samples were incubated at 95°C for 3 min, followed by 12 cycles of (98°C for 20 s, 67°C for 15 s, and 72°C for 4 min), with a final extension of 72°C for 5 min. Samples were purified using Agencourt AMPure XP SPRI beads (Beckman Coulter) and eluted at 25 ml. The final cDNA product was then visualized on an agarose gel to confirm distribution (Figure 2).
Byrne A., Supple M.A., Volden R., Laidre K.L., Shapiro B, & Vollmers C. (2019). Depletion of Hemoglobin Transcripts and Long-Read Sequencing Improves the Transcriptome Annotation of the Polar Bear (Ursus maritimus). Frontiers in Genetics, 10, 643.
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3

Generating DNA Splint via Gibson Assembly

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An ∼200-bp DNA splint to enable Gibson Assembly (20 (link)) circularization of cDNA was amplified from Lambda DNA using KAPA HiFi Readymix 2x (KAPA) (95 °C for 3 min, followed by 25 cycles; 98 °C for 20 s, 67 °C for 15 s, and 72 °C for 30 s) using primer Lambda_F_ISPCR (RC) and Lambda_R_NextA (RC) (SI Appendix, Table S3). This generated a double-stranded DNA with matching overlaps to full-length cDNA.
Volden R., Palmer T., Byrne A., Cole C., Schmitz R.J., Green R.E, & Vollmers C. (2018). Improving nanopore read accuracy with the R2C2 method enables the sequencing of highly multiplexed full-length single-cell cDNA. Proceedings of the National Academy of Sciences of the United States of America, 115(39), 9726-9731.
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4

Amplification and Cloning of STARR-Seq Library

Cited 1 time
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We amplified 5 ng of the HSS library with the following primers: STARR-Seq-AG-f and STARR-Seq-AG-r (Supplemental Table 8) using KAPA HiFi 2x Readymix (Kapa Biosystems) with a 65 °C annealing temperature and 30 s extension. These primers amplify both candidate enhancers and previously assigned degenerate barcodes and add homology arms to the ORI vector (Addgene 99296)22 (link). We followed the reaction in real time with Sybr Green (Thermo fisher scientific) and stopped the reaction before plateauing, at 13 cycles. We gel extracted the amplicon on a 1.5% agarose gel as described above. We then cloned the library in a 2:1 molar excess into 100 ng of the hSTARR-seq_ORI vector (addgene ID:99296), linearized with AgeI and SalI, using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB), following the manufacturer’s protocol. We then transformed 10-beta electrocompetent cells (NEB C3020) with the plasmids in duplicate following the manufacturer’s protocol, along with a no insert negative control. We pooled the two positive transformations during recovery, plated 15 μL to estimate complexity and grew the remainder of the culture in 100 mL LB+Amp. The following day, we estimated the complexity as >500K and extracted the plasmid using the ZymoPURE II Plasmid Midiprep Kit (Zymo Research).
Klein J.C., Agarwal V., Inoue F., Keith A., Martin B., Kircher M., Ahituv N, & Shendure J. (2020). A systematic evaluation of the design and context dependencies of massively parallel reporter assays. Nature methods, 17(11), 1083-1091.
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5

Nucleic Acid Extraction and Purification

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Plasmid DNA was prepared using Monarch Plasmid Miniprep Kit (cat# T1010, New England BioLabs, Ipswich, MA, USA). Genomic DNA was prepared using DNeasy Blood & Tissue Kit (cat# 69504, Qiagen, Hilden, Germany). When necessary, cDNA was purified using Monarch PCR & DNA Cleanup Kit (cat# T1030, New England BioLabs, Ipswich, MA, USA). PCR was performed using either KAPA HIFI 2X ready mix (cat# KK2602, KAPA Biosystems, Wilmington, MA, USA).
Vaillancourt M., Limsuwannarot S.P., Bresee C., Poopalarajah R, & Jorth P. (2021). Pseudomonas aeruginosa mexR and mexEF Antibiotic Efflux Pump Variants Exhibit Increased Virulence. Antibiotics, 10(10), 1164.
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6

Single-Cell RNA Sequencing Pipeline

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RNA was extracted following the Direct-zol RNA MicroPrep kit (Zymo Research) manufacturer’s instructions. RNA was treated with DNAse I (Ambion) and enriched for mRNA using Dynabeads mRNA DIRECT Purification Kit (Thermo Fisher). cDNA was generated using a custom scaled-down modification of the SMART-seq protocol [36 (link)]. cDNA was synthesized from RNA input using Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific). It was then amplified using Kapa HiFi 2X Ready Mix (Kapa Biosystems) and cleaned using 0.9X Ampure beads (Beckman Coulter). Finally, cleaned cDNA samples were tagmented and indexed using the Nextera XT DNA Library Prep Kit (Illumina). Library size and tagmentation were confirmed using the TapeStation HS D1000 kit (Agilent). Libraries were pooled in equal molarity and sequenced with the NextSeq 550 High-Output kit (Illumina) with paired-end 75 bp reads.
Brandt M., Kim-Hellmuth S., Ziosi M., Gokden A., Wolman A., Lam N., Recinos Y., Daniloski Z., Morris J.A., Hornung V., Schumacher J, & Lappalainen T. (2021). An autoimmune disease risk variant: A trans master regulatory effect mediated by IRF1 under immune stimulation?. PLoS Genetics, 17(7), e1009684.
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7

Amplification and Cloning of STARR-Seq Library

Cited 1 time
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We amplified 5 ng of the HSS library with the following primers: STARR-Seq-AG-f and STARR-Seq-AG-r (Supplemental Table 8) using KAPA HiFi 2x Readymix (Kapa Biosystems) with a 65 °C annealing temperature and 30 s extension. These primers amplify both candidate enhancers and previously assigned degenerate barcodes and add homology arms to the ORI vector (Addgene 99296)22 (link). We followed the reaction in real time with Sybr Green (Thermo fisher scientific) and stopped the reaction before plateauing, at 13 cycles. We gel extracted the amplicon on a 1.5% agarose gel as described above. We then cloned the library in a 2:1 molar excess into 100 ng of the hSTARR-seq_ORI vector (addgene ID:99296), linearized with AgeI and SalI, using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB), following the manufacturer’s protocol. We then transformed 10-beta electrocompetent cells (NEB C3020) with the plasmids in duplicate following the manufacturer’s protocol, along with a no insert negative control. We pooled the two positive transformations during recovery, plated 15 μL to estimate complexity and grew the remainder of the culture in 100 mL LB+Amp. The following day, we estimated the complexity as >500K and extracted the plasmid using the ZymoPURE II Plasmid Midiprep Kit (Zymo Research).
Klein J.C., Agarwal V., Inoue F., Keith A., Martin B., Kircher M., Ahituv N, & Shendure J. (2020). A systematic evaluation of the design and context dependencies of massively parallel reporter assays. Nature methods, 17(11), 1083-1091.
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8

Efficient DNA Extraction and Amplification

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Plasmid DNA was prepared using a Monarch Plasmid Miniprep Kit (NEB, cat# T1010). Genomic DNA was prepared using the DNeasy Blood & Tissue Kit (Qiagen, cat# 69504). When necessary, cDNA was purified using the Monarch PCR & DNA Cleanup Kit (NEB, cat# T1030). PCR was performed using either KAPA HIFI 2X ready mix (KAPA Biosystem, cat# KK2602) or Expand Long Template PCR System (Sigma, cat# 11681842001). Primers used for each PCR reaction are listed in S4 Table.
Vaillancourt M., Galdino A.C., Limsuwannarot S.P., Celedonio D., Dimitrova E., Broerman M., Bresee C., Doi Y., Lee J.S., Parks W.C, & Jorth P. (2023). A compensatory RNase E variation increases Iron Piracy and Virulence in multidrug-resistant Pseudomonas aeruginosa during Macrophage infection. PLOS Pathogens, 19(4), e1010942.
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9

Single-Cell RNA-seq Library Preparation

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RNA was extracted following the Direct-zol RNA MicroPrep kit (Zymo Research) manufacturer's instructions. RNA was treated with DNAse I (Ambion) and enriched for mRNA using Dynabeads mRNA DIRECT Purification Kit (Thermo Fisher). cDNA was generated using a custom scaled-down modification of the SMART-seq protocol (Picelli et al., 2014) . cDNA was synthesized from RNA input using Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific). It was then amplified using Kapa HiFi 2X Ready Mix (Kapa Biosystems) and cleaned using 0.9X Ampure beads (Beckman Coulter). Finally, cleaned cDNA samples were tagmented and indexed using the Nextera XT DNA Library Prep Kit (Illumina). Library size and tagmentation were confirmed using the TapeStation HS D1000 kit (Agilent). Libraries were pooled in equal molarity and sequenced with the NextSeq 550 High-Output kit (Illumina) with paired-end 75 bp reads.
Brandt M., Kim-Hellmuth S., Ziosi M., Gokden A., Wolman A., Lam N., Recinos Y., Hornung V., Schumacher J., & Lappalainen T. (2020). An autoimmune disease risk variant has a trans master regulatory effect mediated by IRF1 under immune stimulation.
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