Genomic (g)DNA was extracted from parasites for screening purposes by using DNAzol reagent (Invitrogen, ref. 10503027). All PCRs were conducted by using either Q5 Hot Start High-Fidelity DNA polymerase (New England Biolabs, cat. no. M0494S) for cloning purposes, or MangoMix (Meridian Bioscience ref. BIO-25034) for diagnostic/screening purposes. All PCR conditions were designed following recommendations by NEB TM calculator tool. When necessary for downstream applications (Gibson assembly, cloning, sequencing, etc.), PCR products were purified either directly with the DNA clean and concentration kit (Zymo Research, 11-304C) or after gel excision with the Qiaquick gel extraction kit (Qiagen, 28704). Gibson assembly constructions, plasmids, and PCR products from genes/regions of interest in mutant parasites were assessed by Sanger sequencing using the primers listed in Table S3 , and sequences were analyzed by using BioEdit sequence alignment editor version 7.0.5.3 (127 ).
Dna clean and concentration kit
Manufactured by Zymo Research
Sourced in United States
The DNA Clean and Concentration Kit is a laboratory product designed to purify and concentrate DNA samples. It utilizes a silica-based membrane technology to efficiently recover and purify DNA from various sources, including PCR reactions, enzymatic digestions, and other DNA sample preparations. The kit provides a simple and effective method to remove contaminants and concentrate DNA samples for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Dbco peg4 biotin, by Vector Laboratories (4 mentions)
Streptavidin c1 beads, by Thermo Fisher Scientific (4 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (4 mentions)
Nextseq 500 platform, by Illumina (2 mentions)
Q5 hot start high fidelity dna polymerase, by New England Biolabs (1 mentions)
Dnazol reagent, by Thermo Fisher Scientific (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Anti ha antibody, by Abcam (1 mentions)
H3k27ac, by Diagenode (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Hdac1, by Diagenode (1 mentions)
H3k27me3, by Cell Signaling Technology (1 mentions)
Protein a g bead, by Thermo Fisher Scientific (1 mentions)
Proteinase k, by Zymo Research (1 mentions)
Ribomax large scale rna production systems kit, by Promega (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
Nebnext multiplex oligos for index primers set 1, by Illumina (1 mentions)
Dynabeads myone streptavidin c1, by Thermo Fisher Scientific (1 mentions)
Xten platform, by Illumina (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
10 protocols using dna clean and concentration kit
1
Genomic DNA Extraction and PCR Amplification for Parasite Screening
2
Nanoscale Hydroxymethylcytosine Profiling
3
Chromatin Immunoprecipitation and Sequencing Protocol
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Chromatin immunoprecipitation was performed using a previously described protocol (53 (link)). Briefly, 1 million cells were fixed with 1% formaldehyde and sonicated with Covaris E220 or Bioruptor Plus. Sonicated chromatin was then incubated with antibodies against target proteins or histone posttranslational modifications overnight. Protein A/G beads (Thermo Fisher) were added to the sonicated chromatin for 2 hours. After washes with low-salt, high-salt, and LiCl wash buffer, beads were decross-linked with proteinase K (Zymo Research) at 55°C overnight. Pulled-down DNA was extracted by the DNA clean and concentration kit (Zymo Research). Libraries were prepared using the Swift Bioscience Acce-NGS 2G plus kit according to the manufacturer's manual. The libraries were sequenced 150-bp paired-end on HiSeq 2000 by Fulgent Genomics.
Chromatin immunoprecipitation sequencing (ChIP-seq) antibodies used in this study included H3K27ac (Diagenode, Rabbit Polyclonal, C15410196, RRID:AB_2637079), H3K27me3 (Cell Signaling Technology, Rabbit Monoclonal, 9733, RRID:AB_2616029), Pol II S5P (Diagenode, Mouse Monoclonal, C15200007, RRID:AB_2713926), HDAC1 (Diagenode, Rabbit Polyclonal, C15410325, RRID:AB_2921266), anti-FLAG antibody (Sigma-Aldrich, mouse monoclonal, F1804, RRID:AB_262044), and anti-HA antibody (Abcam, Rabbit Polyclonal, ab9110, RRID:AB_307019).
Chromatin immunoprecipitation sequencing (ChIP-seq) antibodies used in this study included H3K27ac (Diagenode, Rabbit Polyclonal, C15410196, RRID:AB_2637079), H3K27me3 (Cell Signaling Technology, Rabbit Monoclonal, 9733, RRID:AB_2616029), Pol II S5P (Diagenode, Mouse Monoclonal, C15200007, RRID:AB_2713926), HDAC1 (Diagenode, Rabbit Polyclonal, C15410325, RRID:AB_2921266), anti-FLAG antibody (Sigma-Aldrich, mouse monoclonal, F1804, RRID:AB_262044), and anti-HA antibody (Abcam, Rabbit Polyclonal, ab9110, RRID:AB_307019).
4
Zika Virus Nanoluc Plasmid Construction
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The ZIKV-Nanoluciferase (Nanoluc) construct ( Fig. 1A ) used in these assays was described previously34 (link). For maintenance and propagation of the plasmid containing the pCCI-SP6-ZIKV-Nanoluc, the E. coli Turbo strain (New England Biolabs) was used.
Complete amplification of the viral genome was performed using a PCR reaction with Phusion High Fidelity (Thermo Fisher) enzyme and the designed primers ZIKV-Forward (5′ CG ATT AAG TTG GGT AAC GCC AGG GT 3′) and ZIKV-Reverse (5′ T AGA CCC ATG GAT TTC CCC ACA CC 3′). The PCR product containing SP6 promoter followed by complete viral cDNA was purified with the DNA clean and concentration kit (Zymo Research). In vitro transcription was performed using the RiboMAX™ Large-scale RNA Production Systems kit (Promega), as instructed by the manufacturers.
Complete amplification of the viral genome was performed using a PCR reaction with Phusion High Fidelity (Thermo Fisher) enzyme and the designed primers ZIKV-Forward (5′ CG ATT AAG TTG GGT AAC GCC AGG GT 3′) and ZIKV-Reverse (5′ T AGA CCC ATG GAT TTC CCC ACA CC 3′). The PCR product containing SP6 promoter followed by complete viral cDNA was purified with the DNA clean and concentration kit (Zymo Research). In vitro transcription was performed using the RiboMAX™ Large-scale RNA Production Systems kit (Promega), as instructed by the manufacturers.
5
Single-cell RNA-seq of Tumor L-S+ NG2+ Cells
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Single‐cell RNA‐seq using FACS‐isolated L−S+ NG2+ cells from tumor tissue cell fraction was conducted according to the modified Smart‐seq2 protocol as previously described [55 (link), 56 (link)]. After cDNA amplification, 94 single cells with different barcodes were pooled for one library. The mixed cDNAs were purified by a DNA Clean and Concentration Kit (D4014, ZYMO, California, USA), followed by removing the small fragments and primers (<200 bp) by 0.8 × AMPure XP beads (A63882, Beckman, California, USA). The 30∼40 ng purified products were performed with 4 cycles of PCR to append the biotin index to the 3’ end of PCR products. The purified PCR products were fragmented into ∼300‐bp using Covaris S220 (Covaris). Dynabeads MyOne Streptavidin C1 (65001, Invitrogen) was used for the biotin enrichment of shared cDNAs. The library was constructed using KAPA Hyper Prep Kits (KK8504, Kapa Biosystems, Wilmington, MA, USA) and NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) (E7335L, NEB, Ipswich, MA, USA). Sequencing was carried out using the Illumina Xten platform as paired‐end 150‐bp reads.
6
Nanoscale Hydroxymethylcytosine Profiling
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Nano-hmC-Seal was performed as previously described in Han et al.60 (link). Briefly, 100 ng genomic DNA were fragmented in Tagmentation buffer at 55°C. Fragmented DNA was purified by Zymo DNA Clean and Concentration Kit. Then, the selective 5hmC chemical labeling was performed in glucosylation buffer (50 mM HEPES buffer pH 8.0, 25 mM MgCl2) containing above fragmented DNA, βGT, N3-UDP-Glc, and incubated at 37°C for 2 hr. After DNA purification in ddH2O, DBCO-PEG4-Biotin (Click Chemistry Tools) was added and incubated at 37°C for 2 hr. The biotin labeled DNA was pulled down by C1 Streptavidin beads (Life Technologies) for 15 min at room temperature. Next, the captured DNA fragments were subjected to PCR amplification using Nextera DNA sample preparation kit. The resulting amplified product was purified by 1.0X AMPure XP beads. Input library was made by direct PCR from fragmented DNA directly without labeling and pull-down. The libraries were quantified by a Qubit fluorometer (Life Technologies) and sequenced on a NextSeq 500 platform (Illumina) in accordance with the manufacturer’s protocol.
7
Single-Cell Transcriptome Amplification and Sequencing
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We followed STRT-seq steps (Islam et al., 2011 (link), 2014 (link)) to perform single-cell transcriptome amplification with a few modifications in the RT and amplification primers (Li et al., 2017 (link); Zhong et al., 2018 (link)). After amplification for 20 cycles, cDNAs labeled with different barcodes were pooled together and purified using the DNA Clean and Concentration Kit (ZYMO, D5044) to remove primer dimers and free primers. Thereafter, we performed a second amplification using biotin-labeled primers containing the Illumina read2 primer sequence and indexes for four cycles. The cDNAs were then fragmented by sonication with the Covaris S220 (ThermoFisher Scientific, 4465653) and the 5′ end of the first strand cDNA was enriched using C1 streptavidin beads (Invitrogen, 65002). A library was constructed using the KAPA Hyper Prep Kit from Illumina (KAPA, KK8505) according to the manufacturer’s instructions. 0.5 G of 150 bp pair-end reads were obtained for each single cell by sequencing on the Illumina Hiseq4000 as performed by Novogene.
8
5hmC Chemical Labeling and Enrichment
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The 5hmC-Seal was performed as previously described in Han et al.30 (link). Briefly, 100 ng genomic DNA were fragmented in Tagmentation buffer at 55 °C. Fragmented DNA was purified by Zymo DNA Clean and Concentration Kit. Then, the selective 5hmC chemical labeling was performed in glucosylation buffer (50 mM HEPES buffer pH 8.0, 25 mM MgCl2) containing above fragmented DNA, βGT, N3-UDP-Glc, and incubated at 37 °C for 2 h. After DNA purification in ddH2O, DBCO-PEG4-Biotin (Click Chemistry Tools) was added and incubated at 37 °C for 2 h. The biotin-labeled DNA was pulled down by C1 Streptavidin beads (Life Technologies) for 15 min at room temperature. Next, the captured DNA fragments were subjected to PCR amplification using Nextera DNA sample preparation kit. The resulting amplified product was purified by 1.0X AMPure XP beads. Input library was made by direct PCR from fragmented DNA directly without labeling and pull-down. The libraries were quantified by a Qubit fluorometer (Life Technologies) and sequenced on NextSeq 500.
9
5hmC Profiling Protocol for Genomic DNA
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5hmC profiling was performed as described(Han et al., 2016 (link)). Briefly, 100 ng genomic DNA were fragmented in Tagmentation buffer at 55°C. Fragmented DNA was purified by Zymo DNA Clean and Concentration Kit. Then, the selective 5hmC chemical labeling was performed in glucosylation buffer (50 mM HEPES buffer pH 8.0, 25 mM MgCl2) containing above fragmented DNA, βGT, N3-UDP-Glc, and incubated at 37°C for 2 hr. After DNA purification in ddH2O, DBCO-PEG4-Biotin (Click Chemistry Tools) was added and incubated at 37°C for 2 hr. The biotin labeled DNA was pulled down by C1 Streptavidin beads (Life Technologies) for 15 min at room temperature. Next, the captured DNA fragments were subjected to PCR amplification using Nextera DNA sample preparation kit. The resulting amplified product was purified by 1.0X AMPure XP beads. Input library was made by direct PCR from fragmented DNA directly without labeling and pull-down. The libraries were quantified by a Qubit fluorometer (Life Technologies) and sequenced on NextSeq 500 PE42.
Adaptors and low quality nucleotides were trimmed from raw sequencing reads by Trim_Galore, and bowtie was used to align clean reads to mm9 reference genome. Peak calling was performed by MACS1.4. DESeq2 was further used to calculate differentially hydroxymethylated genes and regions.
Adaptors and low quality nucleotides were trimmed from raw sequencing reads by Trim_Galore, and bowtie was used to align clean reads to mm9 reference genome. Peak calling was performed by MACS1.4. DESeq2 was further used to calculate differentially hydroxymethylated genes and regions.
10
RNAi-based Gene Silencing in Mosquitoes
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RNAi experiments were performed as previously described (Kwon et al., 2017 (link); Kwon and Smith, 2019 (link); Reynolds et al., 2020 (link); Smith et al., 2016 (link); Smith et al., 2015 (link)). T7 primers for lozenge (Lz; AGAP002506) were designed using the E-RNAi web application (http://www.dkfz.de/signaling/e-rnai3/idseq.php ) and listed in Supplementary file 7 . T7 templates for dsRNA synthesis were prepared from amplified cDNA from 4 day old whole naïve mosquitoes. PCR amplicons were purified using the DNA Clean and Concentration kit (Zymo Research), and dsRNAs were synthesized using the MEGAscript RNAi kit (Life Technologies). Subsequent dsRNA targeting GFP (control) or Lz was resuspended in nuclease-free water to 3 µg/µl after ethanol precipitation. Injections were performed in 3- to 4-day-old cold anesthetized mosquitoes by intrathoracic injection with 69 nl (~200 ng) of dsRNA per mosquito using a Nanoject III. The effects of gene silencing were measured at 3 days post-injection in whole mosquitoes (n=15) by qRT-PCR as previously described (Kwon and Smith, 2019 (link)).
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