We extracted RNA using the Direct‐zol RNA MiniPrep Kit (R2050, ZymoResearch) following a modification of the manufacturer's instructions (Zhang et al., 2013 ). To obtain sufficient cDNA for sequencing, we amplified each antennal RNA sample independently using the NuGEN Ovation V.2 kit (M01206, NuGEN Technologies), following the manufacturer's instructions. All samples were sequenced on the Illumina HiSeq 2500 platform with a 150 bp paired‐end protocol (at the High Throughput Genomics Core at the Biodiversity Research Center, Academia Sinica, Taiwan).
Nugen ovation v2 kit
Manufactured by Tecan
Sourced in United States
The Nugen Ovation V2 kit is a lab equipment product designed for RNA amplification. It provides a method for generating high-quality amplified cDNA from low input RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Mirneasy mini kit, by Qiagen (6 mentions)
Next ultra rna library prep kit, by New England Biolabs (6 mentions)
Tapestation, by Agilent Technologies (6 mentions)
Nextera protocol, by Illumina (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Nextseq 500, by Illumina (4 mentions)
Tapestation 2200, by Agilent Technologies (4 mentions)
Bcl2fastq2 conversion software v2, by Illumina (4 mentions)
S2 system, by Covaris (4 mentions)
Truseq dna sample kit, by Illumina (4 mentions)
Hiseq 4000 sequencer, by Illumina (4 mentions)
Bluepippin, by Sage Science (3 mentions)
Hiseq 2500, by Illumina (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
2100 expert software, by Agilent Technologies (2 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Novaseq sequencer, by Illumina (1 mentions)
Hiseq 2500 instrument, by Illumina (1 mentions)
15 protocols using nugen ovation v2 kit
1
Antennal RNA Extraction and Sequencing
2
RNA Purification, Library Prep, and Sequencing
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We purified total RNA using the miRNeasy Mini kit (Qiagen, cat#217004) and analyzed RNA concentration and integrity with TapeStation (Agilent) and Qubit. All samples showed RNA integrity numbers higher than 8.4. We then generated cDNA using the Nugen Ovation V2 kit (Nugen), fragmented the cDNA using a Covaris sonicator, and generated sequencing libraries using the Next Ultra RNA Library Prep kit (New England Biolabs) with 9–10 cycles of PCR amplification. We sequenced the libraries with Illumina HiSeq 4,000 and NovaSeq sequencers and obtained 16.3 ± 5.7 million (mean ± standard deviation) 50 bp and 100 bp single-endreads per sample.
3
Viral Metagenomics Sequencing Pipeline
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RNA of six individual viral isolates extracted from supernatants of infected cells was provided for MDS. The investigators preparing the MDS libraries and performing the bioinformatics analysis were blinded to the presence or absence of any known microbial contaminants and also to the strain and isolate identity of each virus. The six viral isolates were LACV strain H78, EBOV, rCDVRI, rMVKS, rHRSVB05, and the VSVIN strain. Samples were processed for MDS analysis as previously described (9 (link)). Samples were randomly amplified to double-stranded cDNA using the NuGEN Ovation v.2 kit (NuGEN, San Carlos, CA), and MDS libraries were constructed using the Nextera protocol (Illumina, San Diego, CA). Each library was dual indexed in the same manner as described above. Samples were pooled into a single library before library size and concentration were determined using the Blue Pippin (Sage Science, Beverly, MA) and Kapa universal quantitative PCR (qPCR) kit (Kapa Biosystems, Woburn, MA), respectively. Samples were sequenced on an Illumina HiSeq 2500 instrument using 150-nt paired-end sequencing. The paired-end sequences were analyzed for microbes using a rapid computational pipeline for microbial detection.
4
Microglial RNA Sequencing Protocol
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RNA clean-up from isolated microglial cells (50,000), TRAP samples and 5% of the unbound fractions from TRAP samples was performed using RNeasy Mini Kit (Qiagen) following manufacturer’s instructions. RNA integrity was assayed using an RNA Pico chip on a Bioanalyzer 2100 (Agilent, Santa Clara, CA), and only samples with RIN>9 were considered for subsequent analysis. Double-stranded cDNA was generated from 1-5 ng of RNA using Nugen Ovation V2 kit (NuGEN, San Carlos, CA) following manufacturer’s instructions. Fragments of 200 bp were obtained by sonicating 500 ng of cDNA per sample using the Covaris-S2 system (Duty cycle: 10%, Intensity: 5.0, Bursts per second: 200, Duration: 120 seconds, Mode: Frequency sweeping, Power: 23W, Temperature: 5.5°C - 6°C, Covaris Inc., Woburn, MA). Subsequently, these fragments were used to produce libraries for sequencing by TruSeq DNA Sample kit (Ilumina, San Diego, CA, USA) following manufacturer’s instructions. The quality of the libraries was assessed by 2200 TapeStation (Agilent). Multiplexed libraries were directly loaded on NextSeq 500 (Ilumina) with High Output single read sequencing for 75 cycles. Raw sequencing data was processed using Illumina bcl2fastq2 Conversion Software v2.17.
5
Microglial RNA Sequencing Protocol
6
Astrocyte Transcriptome Profiling under EGFR and P53 Inhibition
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We harvested astrocytes purified from P2 mouse cerebral cortex and cultured in serum‐free conditions for 2, 7, and 14 days for RNA‐seq. To inhibit EGFR signaling, we added 0.05 μM of the EGFR inhibitor PD168393 at 2 div and harvested cells at 3 div. To inhibit P53, we added 5 μM of the P53 inhibitor Pifithrin‐α at 2 div and harvested cells at 4 div. We used 2–3 biological replicates per condition. We purified total RNA using the miRNeasy Mini kit (Qiagen Cat# 217004) and analyzed RNA concentration and integrity with TapeStation (Agilent) and Qubit. All samples have RNA integrity numbers higher than 7. We then generated cDNA using the Nugen Ovation V2 kit (Nugen), fragmented cDNAs using the Covaris sonicator, and generated sequencing libraries using the Next Ultra RNA Library Prep kit (New England Biolabs) with 10 cycles of PCR amplification. We sequenced the libraries with the Illumina HiSeq 4,000 sequencer and obtained 12.9 ± 2.8 million (mean ± standard deviation [SD]) 50 bp single‐end reads per sample.
7
Unbiased Metagenomic Sequencing of CSF
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On day 11 of the patient's hospitalization, a sample of CSF was submitted for unbiased MDS under a research protocol for the identification of potential pathogens approved by the institutional review boards of San Francisco General Hospital (SFGH) and the University of California San Francisco (UCSF). Samples were processed for MDS analysis, as previously described.1 Briefly, total RNA was extracted from CSF. Samples were randomly amplified to double‐stranded cDNA using the NuGEN Ovation v.2 kit (NuGEN, San Carlos, CA) for low nucleic acid content samples, and MDS libraries were constructed using the Nextera protocol (Illumina, San Diego, CA), as previously described.1 Library size and concentration were determined using the Blue Pippin (Sage Science, Beverly, MA) and Kapa Universal quantitative PCR kit (Kapa Biosystems, Woburn, MA), respectively. Samples were sequenced on an Illumina HiSeq 2500 instrument using 135/135‐base‐pair (bp) paired‐end sequencing. The paired‐end sequences were analyzed for pathogens using a rapid computational pipeline for pathogen detection, as described below.
8
Metagenomic Detection of Pathogens in CSF
9
RNA-Seq Analysis of TRAP Samples
10
RNA-Seq Library Preparation Protocol
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Total RNA was extracted using the miRNeasy Mini kit (Qiagen cat #217004). The concentrations and integrities of the RNA were measured using TapeStation (Agilent) and Qubit. Sixty ng total RNA from each sample was used for library preparation. cDNA was generated using the Nugen Ovation V2 kit (Nugen) and fragmented using the Covaris sonicator. Sequencing libraries were prepared using the Next Ultra RNA Library Prep kit (New England Biolabs) with 12 cycles of PCR amplification. An Illumina HiSeq 4000 sequencer was used to sequence these libraries and each sample had an average of 19.1 ± 2.9 million 50 bp single-end reads.
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