The cDNA library was established using the RNA-Seq Library Preparation Kit (Illumina) following the manufacturer’s protocol. In brief, poly-(A) mRNA was separated from 5 μg of total RNA using the Oligo d(T) magnetic beads. Then, after the fragmentation buffer was added, the mRNA was fragmented into small pieces under an elevated temperature. Using the mRNA fragments as templates, the first-strand cDNA was synthesized with 6-base random primers. Next, the second-strand cDNA was synthesized using DNA polymerase I and RNase H. The obtained cDNAs were end-repaired by polymerase and ligated with “A-tailing” base adaptors. Target fragments were selected by magnetic beads for PCR amplification to construct the final cDNA library and the final double-stranded cDNA samples were examined by agarose electrophoresis. After the library was quantified using Qubit 2.0 (Illumina), sequencing was performed on an Illumina HiSeq 2500 sequencing platform.
Qubit 2
Manufactured by Illumina
Sourced in United States
The Qubit 2.0 Fluorometer is a compact and user-friendly instrument designed for the quantification of nucleic acids and proteins. It utilizes fluorescence-based detection to provide accurate and sensitive measurements. The Qubit 2.0 is capable of analyzing a wide range of sample types, making it a versatile tool for various applications in the laboratory.
Automatically generated - may contain errors
Lab products found in correlation
Miseq reagent kit v3 600 cycle, by Illumina (2 mentions)
Nebnext ultra 2 directional rna second strand synthesis module, by New England Biolabs (2 mentions)
Respiratory virus oligo panel, by Illumina (2 mentions)
Nebnext rna first strand synthesis module, by New England Biolabs (2 mentions)
2100 bioanalyzer, by Illumina (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rna seq library preparation kit, by Illumina (1 mentions)
Hiseq 2500 sequencing platform, by Illumina (1 mentions)
Paired end sample preparation kit, by Illumina (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 tapestation, by Agilent Technologies (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Rna nano 6000 assay kit, by Agilent Technologies (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
6 protocols using qubit 2
1
RNA-Seq Library Preparation and Sequencing
2
SARS-CoV-2 Sequencing from Clinical Samples
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A set of 126 isolates (Ct range 11.29–26.39; see Table S1 ) and one positive (pc-Illumina) and one negative (nc-Illumina) control were transcribed into ds cDNA using NEBNext® RNA First Strand Synthesis module and NEBNext UltraII Directional RNA Second Strand Synthesis module (New England Biolabs; Ipswich, MA, USA) following the manufacturer’s protocol.
Libraries were prepared using Nextera Flex for Enrichment (pre-enrichment part of manufacturer’s protocol;https://emea.support.illumina.com/sequencing/sequencing_kits/illumina-dna-prep-with-enrichment/documentation.html (accessed on 30 July 2021)). Next, libraries were combined in twelve plexes by 7–11 samples (including controls) based on the Ct values (in order to minimise the Ct difference in samples within each plex) and enriched using the Respiratory Virus Oligo Panel (Illumina, San Diego, CA, USA), following the manufacturer’s protocol (https://www.illumina.com/content/dam/illumina-marketing/documents/products/appnotes/coronavirus-enrichment-product-list-1270-2020-004.pdf accessed on 30 July 2021).
Enriched plexes were equally pooled based on evaluation by Qubit 2.0 and Bioanalyzer 2100 and sequenced on the MiSeq platform in a run with configuration 2 × 176 bp using MiSeq Reagent Kit v3 (600 cycle) (Illumina, San Diego, CA, USA).
Libraries were prepared using Nextera Flex for Enrichment (pre-enrichment part of manufacturer’s protocol;
Enriched plexes were equally pooled based on evaluation by Qubit 2.0 and Bioanalyzer 2100 and sequenced on the MiSeq platform in a run with configuration 2 × 176 bp using MiSeq Reagent Kit v3 (600 cycle) (Illumina, San Diego, CA, USA).
3
Transcriptome Analysis of w1118 and dIlp8-/- Flies
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Total RNAs were extracted from three replicates group of 3 days old virgin females of w1118 and dIlp8−/− stocks. The quantity and quality of RNA have been assessed by Nanodrop, Qubit 2.0 and Agilent 2100. Magnetic beads with oligo (dT) were performed to enrich Poly (A) mRNA from total RNA. The cDNA library was constructed using the Paired-End Sample Preparation Kit (Illumina Inc., San Diego, CA, United States) followed the manufacturer’s instructions. To ensure quality control, Qubit 2.0, Agilent 2100 and quantitative Real-Time PCR were performed, and then the transcriptome sequencing was carried out using IlluminaHiSeq 6000 with PE100 approach (Biomarker Technology Corporation, Beijing, China). The clean reads were filtered from raw data by removing adaptor sequence, primer reads and low-quality bases. We use the longest isoform to represent a gene for downstream analysis. The reads of each sample were mapped to the reference genome of D. melanogaster. The gene abundance was represented by PKM value. DEG screening in sample group was conducted with the DEGseq package. False discovery rate (FDR) value ≤ .01 and fold change (FC) ≥ 2 in the Benjamini and Hochberg method were chosen as DEGs.
4
RNA-seq Analysis of Mouse Transcriptome
5
Respiratory Virus Enrichment Sequencing
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A set of 35 isolates (Ct values 11.29–40, Supplementary Table S1 )—4 positive (pc1, pc2, pc3, pc4) and 1 negative (nc2) controls—was transcribed into ds cDNA using NEBNext® RNA First Strand Synthesis Module and NEBNext® Ultra™ II Directional RNA Second Strand Synthesis Module (New England Biolabs; Ipswich, MA, USA) following manufacturer’s protocol.
Libraries were prepared using Nextera Flex for Enrichment (pre-enrichment part of manufacturer’s guide;https://emea.support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/illumina_prep/illumina-dna-prep-with-enrichment-reference-1000000048041-05.pdf ). Next, libraries were combined within 8 plexes by 5 samples each based on Ct values (see Supplementary Table S1 ) and enriched using the Respiratory Virus Oligo Panel (Illumina, San Diego, CA, USA, following the manufacturer’s protocol (https://www.illumina.com/content/dam/illumina-marketing/documents/products/appnotes/coronavirus-enrichment-product-list-1270-2020-004.pdf ).
Enriched plexes were equally pooled based on evaluation by Qubit 2.0 and Bioanalyzer 2100 and sequenced on the MiSeq platform using MiSeq Reagent Kit v3 (600 cycle) (Illumina, San Diego, CA, USA).
Libraries were prepared using Nextera Flex for Enrichment (pre-enrichment part of manufacturer’s guide;
Enriched plexes were equally pooled based on evaluation by Qubit 2.0 and Bioanalyzer 2100 and sequenced on the MiSeq platform using MiSeq Reagent Kit v3 (600 cycle) (Illumina, San Diego, CA, USA).
6
Panda Sperm RNA Extraction and Sequencing
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Prior to total RNA extraction, semen was treated with 0.5% Triton X-100 according to previous study to eliminate somatic cell contamination [25 (link)]. For each panda sample (n = 5), total RNA extraction of fresh and frozen-thawed sperm was performed with Trizol LS Reagent (Invitrogen, Carlsbad, CA). RNA concentration, purity and RNA integrity were measured using the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System (Agilent Technologies, Santa Clara, CA, USA), respectively. Then, miRNA libraries were constructed using NEBNext Ultra-small RNA Sample Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) according to manufacturer’s instructions. The quality and yield after sample preparation were determined with Agilent 2100 Tape Station and Qubit 2.0, and the RNA was sequenced on an Illumina Hiseq 2500 platform.
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