3 μg of total RNA per sample was used as input material for the RNA sample preparation. Beads with oligo (dT) were used to isolate poly(A) mRNA from total RNA. RNA sequencing libraries were constructed from these mRNA using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, USA). Briefly, the Elution 2-Frag-Prime (94 °C for 8 minutes, 4 °C hold) was used to elute, fragment and prime the mRNA with Elute, Prime, Fragment Mix (Illumina). First strand cDNA synthesis was performed with First Strand Master Mix and SuperScript II mix (ratio: 1ul SuperScript II/7ul First Strand Master Mix) (Invitrogen). The second strand was synthesized with Second Strand Master Mix (Illumina) and Ampure XP beads (Illumina) were used to separate the double-stranded (ds) cDNA from the 2nd strand reaction mix. After end repair and the addition of a 3′-dA overhang, the cDNA was ligated to Illumina PE adapter oligo mix (Illumina), and size-selected for 350 ± 20 bp fragments by gel purification. After 15 cycles of PCR amplification, the 350 bp paired-end libraries were sequenced using the paired-end sequencing module (100 bp at each end) of the Illumina HiSeq 2000 platform.
First strand master mix
Manufactured by Thermo Fisher Scientific
Sourced in United States
First Strand Master Mix is a ready-to-use solution for the reverse transcription of RNA to create first-strand cDNA. It contains all the necessary components, including reverse transcriptase, primers, and dNTPs, to perform this essential step in gene expression analysis and other applications.
Automatically generated - may contain errors
Lab products found in correlation
Ribo zero magnetic kit, by Illumina (3 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (3 mentions)
Superscript 2, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Agencourt rnaclean xp beads, by Beckman Coulter (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Hiseq x ten, by Illumina (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Paired end adapter, by Illumina (1 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Thermo Fisher Scientific (1 mentions)
Sequencing system, by Illumina (1 mentions)
Agilent 2100 bioanalyzer instrument, by Agilent Technologies (1 mentions)
Hiseqtm 2500 platform, by Illumina (1 mentions)
Ribo zero magnetic gold kit, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Truseq rna sample prep kit v2, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Illumina (1 mentions)
12 protocols using first strand master mix
1
Illumina RNA-Seq Library Preparation
2
RNA-seq Library Preparation for Stem Segments
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RNAs from the five stem segments, bark and xylem, were used for RNA-seq library construction and sequencing, and three biological replicates were performed for each sample. Briefly, total RNA from each sample was processed with DNase I, and mRNA was enriched with oligo (dT) magnetic beads and fractured into short fragments through fragmentation buffer. First-strand cDNA was generated using First Strand Master Mix and SuperScript II reverse transcription (Invitrogen). With DNA polymerase I and RNaseH, the second-strand cDNA was further synthesized and purified through AMPure XP beads (Beckman, United States). After end repairing, poly (A) tail was added, and adaptor ligation was also done. Subsequently, cDNA fragments were enriched by PCR amplification. The final library was evaluated using the Qubit 2.0, Agilent 2,100 bioanalyzer instrument, and qRT-PCR. The qualified libraries were sequenced on the Illumina HiSeq X Ten platform. After sequencing, raw reads were further filtered to generate clean reads by removing adaptors, low-quality reads and reads containing more than 10% unknown nucleotides. The Q30 and GC contents of clean reads were calculated based on quality control analysis.
3
Illumina Sequencing of mRNA
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Illumina (San Diego, CA) sequencing was performed at the Beijing Genomic Institute (BGI-Shenzhen, China) following the manufacturer’s instructions. Briefly, rRNA was depleted using the Ribo-Zero Magnetic kit (Epicentre, Madison, WI). The mRNA was fragmented into small pieces (130-170 nt) and purified with Agencourt RNAClean XP Beads (Beckman Coulter Genomics). These cleaved mRNA fragments served as a template for the synthesis of the first-strand cDNA using First Strand Master Mix and SuperScript II reverse transcriptase (Invitrogen). Second-strand cDNA was then synthesized using SuperScript Double-Stranded cDNA Synthesis kit (Invitrogen, Camarillo, CA). These cDNA fragments were further processed with end reparation and poly (A) addition. The repaired cDNA fragments were 3’-adenylated. Illumina’s paired-end adapters were ligated to the ends of these 3’-adenylated cDNA fragments. The products from this ligation reaction were electrophoresed to select a suitable size range of fragments for PCR amplification. Finally, cDNA libraries with a fragment length range of 200 bp were constructed and sequenced on an Illumina HiSeqTM 2000 (TruSeq SBS KIT-HS V3, Illumina) platform43 (link).
4
Illumina Sequencing Library Preparation from Plant Leaf RNA
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Illumina sequencing libraries were prepared as previously described (Li et al., 2021 (link)). In brief, a Ribo-Zero™ Magnetic Kit (Plant Leaf) (Epicentre) was used to treat approximately 1 µg of total RNA per sample to deplete the rRNA. The retrieved RNA was interrupted by adding the First Strand Master Mix (Invitrogen, Carlsbad, CA, USA). First-strand cDNA was generated by reverse transcription with random primers, and second-strand cDNA was synthesized. The synthetic cDNA was subjected to end-repair and was 3’ adenylated. Adapters were attached to the ends of the 3’-adenylated cDNA fragments. Several rounds of PCR amplification were performed using the PCR Primer Cocktail and PCR Master Mix for enrichment of cDNA fragments. Then, the PCR products were purified using Ampure XP Beads. The final library was qualified and quantified. The qualified library was double-sequenced on the BGISEQ platform (Beijing Genomic Institute [BGI], Shenzhen, China).
5
RNA Extraction and Transcriptome Sequencing
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Total RNA was extracted from EAT samples using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The RNA quality and quantity were evaluated using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA) and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA).
The Ribo-Zero Magnetic Kit (Epicentre) was used to process approximately 1 μg of total RNA in each sample to deplete rRNA. The First Strand Master Mix (Invitrogen) was added to fragment the retrieved RNA. The synthesized cDNA was end-repaired and then 3′adenylated. The ends of these 3′adenylated cDNA fragments were connected using adaptors. PCR Primer Cocktail and PCR Master Mix were used for PCR amplification to enrich cDNA fragments. The PCR product was then purified using Ampure XP beads. Two methods were used for the qualification and quantification of the final library: using Agilent 2100 bioanalyzer to check the size distribution of fragments, and using real-time quantitative PCR (RT-qPCR) (TaqMan Probe) for the library quantification. Qualified libraries were sequenced on the Hiseq 4000 or Hiseq X-ten platform (BGI, Shenzhen, China).
The Ribo-Zero Magnetic Kit (Epicentre) was used to process approximately 1 μg of total RNA in each sample to deplete rRNA. The First Strand Master Mix (Invitrogen) was added to fragment the retrieved RNA. The synthesized cDNA was end-repaired and then 3′adenylated. The ends of these 3′adenylated cDNA fragments were connected using adaptors. PCR Primer Cocktail and PCR Master Mix were used for PCR amplification to enrich cDNA fragments. The PCR product was then purified using Ampure XP beads. Two methods were used for the qualification and quantification of the final library: using Agilent 2100 bioanalyzer to check the size distribution of fragments, and using real-time quantitative PCR (RT-qPCR) (TaqMan Probe) for the library quantification. Qualified libraries were sequenced on the Hiseq 4000 or Hiseq X-ten platform (BGI, Shenzhen, China).
6
RNA-seq Library Preparation for Bat Tissue
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Total RNA was extracted from the BAT using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The total RNA from each sample was treated with Ribo-Zero™ Magnetic Kit (Epocentre) to deplete any ribosomal RNA. The retrieved RNA was fragmented by adding First Strand Master Mix (Invitrogen). First-strand cDNA was generated using random primers reverse transcription, followed by second-strand cDNA synthesis in which the RNA template was removed and dTTP was replaced by dUTP. The incorporation of dUTP prevents the second strand of cDNA from being amplified in the subsequent process because the polymerase cannot cross the dUTP site on the template during extension. The double-stranded cDNA product was then ligated with an "A" base and a linker. The ligation product was amplified, and the final 10 cDNA sequencing libraries were obtained after purification (three biological replicates of the OP and OR groups, and four biological replicates of ND). The final library was qualified and quantitated using an Agilent 2100 Bioanalyzer to check the distribution of the size of fragments (quality), and qPCR to quantify the library. The qualified libraries were pair-end sequenced on a HiSeq X Ten platform (BGI-Shenzhen, China).
7
RNA-seq Library Preparation Protocol
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Ribo‐Zero Magnetic Kit (Plant Leaf, Madison,Wisconsin, USA) was applied to treat total RNA for depleting rRNA. The First Strand Master Mix (Invitrogen) was used to fragment the retrieved RNA. After library construction, Ampure XP Beads were used to purify the polymerase chain reaction (PCR) products. The average molecule length was determined using the Agilent 2100 bioanalyzer instrument, and the libraries were quantified by quantitative real‐time polymerase chain reaction (qRT‐PCR) and sequenced pair end on the Illumina sequencing system.
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Ribo‐Zero Magnetic Kit (Plant Leaf, Madison,Wisconsin, USA) was applied to treat total RNA for depleting rRNA. The First Strand Master Mix (Invitrogen) was used to fragment the retrieved RNA. After library construction, Ampure XP Beads were used to purify the polymerase chain reaction (PCR) products. The average molecule length was determined using the Agilent 2100 bioanalyzer instrument, and the libraries were quantified by quantitative real‐time polymerase chain reaction (qRT‐PCR) and sequenced pair end on the Illumina sequencing system.
8
RNA-Seq Library Preparation and Sequencing
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RNA integrity was measured using Bioanalyzer 2100 (Agilent, Santa Clara, CA, United States) and rRNA was removed from 1 mg of total RNA with Ribo-Zero Magnetic Gold Kit (Epicentre Biotechnologies, Madison, WI, United States). To construct the RNA-Seq library, TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA, United States) was used. rRNA-depleted RNA was fragmented into small pieces using Elute Prime Fragment Mix. First-strand cDNA was synthesized with First Strand Master Mix and Super Script II reverse transcriptase (Invitrogen, Carlsbad, CA, United States). Following purification by Agencourt RNAClean XP beads (Beckman Coulter, CA, United States), the second-strand cDNA library was synthesized using Second Strand Master Mix and dATP, dGTP, dCTP, dUTP mix. Purified fragmented cDNA was end repaired (30 min at 37°C) prior to ligating sequencing adapters. Amplified RNA-Seq libraries were purified by using AMPureXP Beads. The clustering of the index-coded samples was performed on a cBot Cluster Generation System following to the manufacturer’s instructions, and the sequencing was performed using the Illumina Hiseq TM 2500 platform with pair-end 150 base reads.
9
RNA-seq Profiling of Developmental Stages
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Qualified RNA samples of four individuals at the same age were equally pooled together to form three RNA groups: 1 d, 14 d, and 28 d. Approximately 1 μg of total RNA per sample was treated with the Ribo-Zero™ Magnetic Kit (Epicenter) to deplete rRNA. The retrieved RNA was fragmented by adding First Strand Master Mix (Invitrogen). First-strand cDNA was generated using random primer reverse transcription, followed by second-strand cDNA synthesis. The synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adapters were ligated to the ends of these 3' adenylated cDNA fragments. Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix were performed to enrich the cDNA fragments. Then, the PCR products were purified with Ampure XP Beads. The constructed RNA libraries were quality checked with an Agilent 2100 Bioanalyzer and then sequenced using an Illumina sequencer.
10
Transcriptome Analysis of Grape Cultivars
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Extracted RNA was sent to BGI (Shenzhen, China) for transcriptome library construction. In this process, RNA was treated with a Ribo-Zero™ Magnetic Kit to degrade rRNA. First-strand cDNA is generated by First Strand Master Mix and Super Script II reverse transcription (Invitrogen). High-throughput sequencing was performed using a HiSeq 2500 instrument. The clean reads generated by high-throughput sequencing were mapped on the grape genome (http://genomes.cribi.unipd.it/grape/ ) using the HISAT software (V2.0.4)62 (link), and the reads mapped on the genome were assembled into transcripts using the stringTie software (V1.0.4)63 (link).
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