For experiments with application of ODNs in mice and adoptive transfer of pDCs, the corneal epithelium was separated from the stroma as described above. RNA was extracted from TGs, dLNs, whole corneas, and corneal stromas with RNeasy Plus Universal Mini kit (QIAGEN, Germantown, MD). For in vitro experiments, cells were collected after 24-h culture and RNA was extracted via SingleShot Cell Lysis (Bio-Rad Laboratories, Hercules, CA). cDNA was synthetized using iScript cDNA synthesis kit (Bio-Rad Laboratories). For samples obtained from in vitro culture and adoptive transfer studies, cDNA was pre-amplified using SsoAdvanced PreAmp Supermix (Bio-Rad Laboratories) according to the manufacturer’s instructions. For single cell qRT-PCR, 100 corneal GFP+ pDCs, naïve splenic pDCs, and macrophages were lyzed via REPLI-g Cell WGA & WTA kit (QIAGEN) according to manufacturer’s instructions. qRT-PCR was carried out in triplicates using the SYBR Premix EX TaqII (Takara, Japan) or SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories), and analyzed using a Bio-Rad iCycler iQ thermocycler (Bio-Rad Laboratories). The sequence of primers used is available in Table S1 . Relative fold changes are reported using delta-delta cycle threshold (ΔΔCt) method.
Repli g cell wga wta kit
Manufactured by Qiagen
Sourced in Germany
The REPLI-g Cell WGA & WTA Kit is a laboratory product designed for whole genome amplification (WGA) and whole transcriptome amplification (WTA) from a small number of cells. The kit uses a proprietary technology to generate high-quality, representative amplification of genomic DNA and RNA from limited sample material.
Automatically generated - may contain errors
Lab products found in correlation
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (2 mentions)
Icycler iq thermocycler, by Bio-Rad (2 mentions)
Rneasy plus universal mini kit, by Qiagen (2 mentions)
Singleshot cell lysis, by Bio-Rad (2 mentions)
Ssoadvanced preamp supermix, by Bio-Rad (2 mentions)
Minibest viral rna dna extraction kit, by Takara Bio (1 mentions)
Nebnext ultra dna library prep kit, by New England Biolabs (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Agilent 4200 system, by Agilent Technologies (1 mentions)
Nanodrop one, by Thermo Fisher Scientific (1 mentions)
Novaseq pe150, by Illumina (1 mentions)
T4 polynucleotide kinase, by Thermo Fisher Scientific (1 mentions)
T4 dna polymerase, by Promega (1 mentions)
Klenow dna polymerase, by Illumina (1 mentions)
Truseq dna preparation protocol, by Illumina (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Hiseq 4000 platform, by Illumina (1 mentions)
9 protocols using repli g cell wga wta kit
1
Transcriptional Profiling of Corneal Immune Cells
2
Enrichment and Identification of Fecal RNA Viromes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RNA viruses of enrichment of fecal samples were performed as mentioned above. RNA viruses in samples were extracted by using Magen RNA extraction kits (Magen, China), and the whole genome was amplified with Qiagen kit (REPLI-g Cell WGA & WTA Kit).
Sequencing libraries construction and sequencing method were identical as DNA virome. The bioinformatics analyses were similar as mentioned above. Specifically, Blast software (v2.9.0 +) was used to compare the unique contig with virus database, if the alignment similarity was ≥ 80%, the alignment length was ≥ 500 bp, and e ≤ 1e-5 was defined as virus sequence; if the alignment length was ≥ 100 bp, and e ≤ 1e-5 was defined as suspected virus sequence. The candidate viruses sequences were queried against the NCBI reference genomes using BWA-MEM, NT database using MegaBLAST (e-value cutoff 1E-10), BLASTn (e-value cutoff 1E-10), and NCBI NR database using BLASTx (e-value cutoff 1E-3). Sequences with significant hits were classified as novel viruses based on the taxonomy identity of the best BLAST hit [40 (link), 41 (link)]. Parts of the viruses’ contigs, such as Rotavirus, Gammacoronavirus and Deltacoronavirus were aligned using Blast software to compare their similarity with other references. MEGA 6.0 software was used to build the phylogenetic tree using N-J methods with 1000 bootstraps.
Sequencing libraries construction and sequencing method were identical as DNA virome. The bioinformatics analyses were similar as mentioned above. Specifically, Blast software (v2.9.0 +) was used to compare the unique contig with virus database, if the alignment similarity was ≥ 80%, the alignment length was ≥ 500 bp, and e ≤ 1e-5 was defined as virus sequence; if the alignment length was ≥ 100 bp, and e ≤ 1e-5 was defined as suspected virus sequence. The candidate viruses sequences were queried against the NCBI reference genomes using BWA-MEM, NT database using MegaBLAST (e-value cutoff 1E-10), BLASTn (e-value cutoff 1E-10), and NCBI NR database using BLASTx (e-value cutoff 1E-3). Sequences with significant hits were classified as novel viruses based on the taxonomy identity of the best BLAST hit [40 (link), 41 (link)]. Parts of the viruses’ contigs, such as Rotavirus, Gammacoronavirus and Deltacoronavirus were aligned using Blast software to compare their similarity with other references. MEGA 6.0 software was used to build the phylogenetic tree using N-J methods with 1000 bootstraps.
3
Viral Genome Sequencing and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Virus DNA was extracted using MiniBEST Viral RNA/DNA Extraction Kit (TaKaRa, Dalian, China), and the whole genome was amplified with REPLI-g Cell WGA & WTA Kit (Qiagen, Hilden, Germany).
Sequencing libraries were generated using NEB Next® Ultra™ DNA Library Prep Kit (New England Biolabs, MA, United States) following the manufacturer’s recommendations and index codes were added. The library quality was assessed using the Qubit® dsDNA HS Assay Kit (Life Technologies, Grand Island, NY) and Agilent 4200 system (Agilent, Santa Clara, CA). The libraries were sequenced on an Illumina NovaSeq 6000 and 150 bp paired-end reads were generated. The raw data in this study have been deposited in the GenBank Sequence Read Archive database. The accession number is PRJNA797730.
The raw data processing with SOAPnuke (v1.5.6; Chen et al., 2018 (link)) were conducted to acquire the clean data for subsequent analysis. To remove the host sequence, clean reads were compared with the ribosome database (Silva.132) and host reference genome of Pacific white shrimp (NCBI Project ID: PRJNA438564; Zhang et al., 2019 (link)), respectively, with Burrows-Wheeler Aligner (BWA) software (v0.7.17, parameter: mem –k 30; Li and Durbin, 2009 (link)). The results that comparison length was less than 80% of the length of reads would be filtered, and then removed the host sequence (Li and Durbin, 2009 (link)).
Sequencing libraries were generated using NEB Next® Ultra™ DNA Library Prep Kit (New England Biolabs, MA, United States) following the manufacturer’s recommendations and index codes were added. The library quality was assessed using the Qubit® dsDNA HS Assay Kit (Life Technologies, Grand Island, NY) and Agilent 4200 system (Agilent, Santa Clara, CA). The libraries were sequenced on an Illumina NovaSeq 6000 and 150 bp paired-end reads were generated. The raw data in this study have been deposited in the GenBank Sequence Read Archive database. The accession number is PRJNA797730.
The raw data processing with SOAPnuke (v1.5.6; Chen et al., 2018 (link)) were conducted to acquire the clean data for subsequent analysis. To remove the host sequence, clean reads were compared with the ribosome database (Silva.132) and host reference genome of Pacific white shrimp (NCBI Project ID: PRJNA438564; Zhang et al., 2019 (link)), respectively, with Burrows-Wheeler Aligner (BWA) software (v0.7.17, parameter: mem –k 30; Li and Durbin, 2009 (link)). The results that comparison length was less than 80% of the length of reads would be filtered, and then removed the host sequence (Li and Durbin, 2009 (link)).
4
Whole Transcriptome Amplification and Sequencing of Dengue Virus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA concentration and purity were evaluated using Nanodrop One (NanoDrop Technologies, Wilmington, DE, USA), and RNA integrity was determined using agarose gel electrophoresis. The REPLI-g Cell WGA & WTA Kit (Qiagen, Germany) was used for whole transcriptome amplification to obtain double-stranded cDNA for samples with low concentrations, following the manufacturer’s instructions. Library preparation was performed using the ALFA-SEQ DNA Library Prep Kit kit following the manufacturer’s instructions and sequenced on an Illumina NovaSeq-PE150.
The raw sequencing data were first filtered using Soapnuke (https://github.com/BGI-flexlab/SOAPnuke ) to remove low-quality reads and obtain clean data. Then, clean reads were aligned to a human genome reference using the BWA software (v0.7.17) and filtered to remove host contamination that could affect the subsequent data analysis. The clean reads without host contamination were aligned to different genotypes of dengue sequences obtained from the NCBI database, and the sequence with the highest similarity was determined as the reference genome. Clean reads were aligned to the reference sequence using BWA (v0.7.17) and SamTools (v0.7.17) software to obtain a consensus sequence.
The raw sequencing data were first filtered using Soapnuke (
5
Transcriptional Profiling of Corneal Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For experiments with application of ODNs in mice and adoptive transfer of pDCs, the corneal epithelium was separated from the stroma as described above. RNA was extracted from TGs, dLNs, whole corneas, and corneal stromas with RNeasy Plus Universal Mini kit (QIAGEN, Germantown, MD). For in vitro experiments, cells were collected after 24-h culture and RNA was extracted via SingleShot Cell Lysis (Bio-Rad Laboratories, Hercules, CA). cDNA was synthetized using iScript cDNA synthesis kit (Bio-Rad Laboratories). For samples obtained from in vitro culture and adoptive transfer studies, cDNA was pre-amplified using SsoAdvanced PreAmp Supermix (Bio-Rad Laboratories) according to the manufacturer’s instructions. For single cell qRT-PCR, 100 corneal GFP+ pDCs, naïve splenic pDCs, and macrophages were lyzed via REPLI-g Cell WGA & WTA kit (QIAGEN) according to manufacturer’s instructions. qRT-PCR was carried out in triplicates using the SYBR Premix EX TaqII (Takara, Japan) or SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories), and analyzed using a Bio-Rad iCycler iQ thermocycler (Bio-Rad Laboratories). The sequence of primers used is available in Table S1 . Relative fold changes are reported using delta-delta cycle threshold (ΔΔCt) method.
6
Viral Metagenomic Sequencing from Fecal Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fecal samples were processed using a previously published method [22 (link),23 (link)]. Total RNA was extracted from enriched virus-like particles using a QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). The extracted RNA of all samples was pooled for library construction, followed by amplification using the REPLI-g Cell WGA & WTA Kit (150052; Qiagen, Hilden, Germany). Amplified DNA was randomly fragmented by ultrasound sonication (Covaris M220, Woburn, MA, USA) to produce 800 bp fragments, then sticky ends were repaired and adapters were added using T4 DNA polymerase (M4211, Promega, Madison, WI, USA), Klenow DNA Polymerase (KP810250, Epicentre, Woburn, MA, USA), and T4 polynucleotide kinase (EK0031, Thermo Scientific, Fermentas, GlenBurnie, MD, USA). Each viral sequencing library was prepared following the Illumina TruSeq DNA Preparation Protocol and was sequenced on the HiSeq4000 platform (Illumina, San Diego, CA, USA), with 150 bp paired ends. The library preparation and sequencing process was performed by BGI Tech (Shenzhen, China).
7
RNA Extraction and Sequencing from Mosquito Pools
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from 20 mosquito pools using HiPure Universal RNA Mini Kit (Magen, China) according to manufacturer instructions. RNA quality was evaluated using the Agilent 4200 Bioanalyzer (Agilent Technologies), and all extractions had a qualified RNA integrity score. Ribosomal RNA (rRNA) was removed from purified total RNA using the ALFA-SEQ rRNA Depletion Kit (mCHIP, China) and Ribo-Zero™ Magnetic Gold Kit (Epicentre, USA) according to manufacturer instructions before library construction. Subsequently, double-stranded cDNA was synthesized by reverse transcription using REPLI-g Cell WGA & WTA Kit (150,052; Qiagen, Hilden, Germany). Sequencing libraries were then generated using NEB Next® Ultra™ DNA Library Prep Kit for Illumina® (New England Biolabs, Ipswich, MA, USA), based on manufacturer recommendations, and 150 bp paired-end sequencing of each library was performed on the Illumina NovaSeq6000 platform (Illumina, San Diego, CA, USA). All library preparation and sequencing procedures were performed by Magigene Company (Guangzhou, China).
8
WTA/WGA Amplification for SARS-CoV-2 and HPV
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For random amplification, we used Whole Transcriptome Amplification (WTA) for SARS-CoV-2 samples and Whole Genome Amplification (WGA) for HPV samples from the REPLI-g Cell WGA & WTA Kit (Qiagen, Hilden, Germany). The kit includes five steps including cell lysis, genomic DNA removal for WTA, reverse transcription or genomic DNA repair for WTA and WGA, respectively, ligation of small gDNA or cDNA fragments and random amplification with the phi29 DNA polymerase. We made three modifications of the protocol: firstly, we omitted the cell lysis step because the samples had been lysed with MPLB-buffer. Secondly, the 10 µL input material required in the REPLI-g protocol remained bound to the MGPs from the NA extraction step as recommended by Rostenstierne et al. [15] (link). Thirdly, instead of using the oligo dT primers provided in the kit for the reverse-transcription step, we used 20 µM 5′-phosphorylated random hexamers (P-N6) as described earlier [16] (link).
9
Panvirus Microarray Screening of Tick RNA Viromes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Twenty-five μl of extracted NA from 20 randomly selected tick pools were treated with 4 U TURBO™ DNase (ThermoFisher, Waltham, MA, USA) and enriched for RNA virus using an unpublished I. ricinus tickmodified version of the published protocol (Matranga et al., 2016) (link). Five μl of enriched RNA were used for random whole transcriptome amplification (WTA) using the Repli-g Cell WGA & WTA kit (Qiagen, Hilden, Germany) according to the manufactures instructions. Amplified cDNA was purified using the QiaAmp DNA mini kit (Qiagen, Hilden, Germany) and 1.5 μg of purified cDNA was labelled with Cy5 using the SureTag DNA labelling kit (Agilent, Santa Clara, CA, USA). The labelled DNA was pooled according to the sampling date and tick life-cycle stage and hybridized to a PanVirus microarray (SurePrint G3 Custom Gene Expression Microarray (4 x 180 K) (Agilent, Santa Clara, CA, USA)) containing 60-160 probes for each sequenced virus described in Gen-Bank (2016) . Microarray data was analysed using a simple Excel-based data analysis method as described previously (Erlandsson et al., 2011; (link)Rosenstierne et al., 2014) (link).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!