For ten of the 12 species (all except A. amphitrite and T. j. formosana, for which transcriptome data had already been published), tissue samples (single individual of each species) of the prosoma and base/peduncle (where the cement gland is located) were carefully isolated without contaminating each other. The total RNA of the prosoma and base was extracted using TRIzol® Reagent (Invitrogen, Camarillo, CA) and High Pure RNA Isolation Kit (Roche Applied Science, Germany), respectively. RNA quality assessment was conducted by a Bioanalyzer 2100 with RNA 6000 labchip kit (Agilent Technologies, Santa Clara, CA, USA). cDNA libraries for both parts of all 10 species were prepared using Illumina TruSeq RNA Sample Prep Kits v2 and subsequently sequenced by HiSeq™ 2500 High-Throughput Mode v4 with paired-end 125 base-pair reads located at the High throughput Genomics Core, Biodiversity Research Center, Academia Sinica, Taiwan according to the manufacturer’s instructions (Illumina, San Diego, CA).
Truseq rna sample prep kits v2
Manufactured by Illumina
Sourced in United States
The TruSeq RNA Sample Prep Kits v2 are a set of reagents and consumables designed for the preparation of RNA samples for sequencing on Illumina platforms. The kits provide the necessary components for converting RNA into a library of template molecules suitable for subsequent cluster generation and DNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (6 mentions)
Ribo zero rrna removal kit, by Illumina (5 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Rneasy column, by Qiagen (4 mentions)
Dnase, by Qiagen (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
2100 bioanalyzer, by Agilent Technologies (4 mentions)
Hiseq 2500 high throughput mode v4, by Illumina (3 mentions)
Rna 6000 labchip kit, by Agilent Technologies (3 mentions)
Fast sybr green master mix, by Thermo Fisher Scientific (3 mentions)
Turbo dnase, by Thermo Fisher Scientific (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Improm 2, by Promega (3 mentions)
Agilent 2100 bioanalyser, by Agilent Technologies (3 mentions)
Rneasy plant kit, by Qiagen (3 mentions)
Rnaclean xp, by Beckman Coulter (2 mentions)
Hiseq x10, by Illumina (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
27 protocols using truseq rna sample prep kits v2
1
Transcriptome Analysis of Barnacle Species
2
RNA-seq analysis of Chlorocebus sabeus transcriptome
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Total RNA was extracted using the RNeasy kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. Illumina cDNA libraries were constructed with the TruSeq RNA Sample Prep Kits v2 (Illumina, San Diego, CA, USA) following the manufacturer’s protocol. Sequencing of the cDNA libraries was performed using an Illumina HiSeq2000. Reads were trimmed using the CLC Genomics Workbench 9.4.1 with the following parameters: ‘qualityscores—0.005; trim ambiguous nucleotides—2; remove 5′ terminal nucleotides—1; remove 3′ terminal nucleotides—1; discard reads below length 25’. Trimmed reads were mapped using the RNA-seq mapping algorithm implemented in the CLC Genomics Workbench to the reference Chlorocebus_sabeus genome V 1.1. allowing only unique mapping (length fraction = 1, similarity fraction = 0.95). Differentially expressed genes were inferred using the R package “DESeq2”. FDR < 0.05 and 0.5 < FC < 2 were used as thresholds of statistical significance.
3
RNA-Seq and qRT-PCR Workflow for Lymphoma Analysis
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Total RNA was purified onto RNeasy columns (Qiagen) and treated on-column with DNase (Qiagen). Complementary DNA was produced using the reverse transcriptase ImPromII (Promega, Madison, WI, USA). 10 ng of complementary DNA were used for Real-time RT-PCR reactions with FAST SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA).
RNA-Seq was performed on two biological replicates of four Eμ-myc Trp53KI/Δ lymphomas treated with 100 nM OHT or 1/1000 vol. ethanol for 2 h and three Eμ-myc Arf−/−Trp53+/+ lymphomas treated with 5 μM (−)−Nutlin or 1 μM doxorubicin for 3 h and the corresponding controls (5 μM (+)−Nutlin or 1/1000 vol. H2O, respectively). Total RNA from 107 cells was purified using Trizol (Invitrogen), treated with Turbo DNase (Ambion, Austin, TX, USA) and purified with Agencourt RNA Clean XP (Beckman Coulter, Brea, CA, USA). 5 μg of purified RNA were then treated with Ribozero rRNA removal kit (Epicentre, Madison, WI, USA) and ethanol precipitated. RNA quality and removal of rRNA were checked with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries for RNA-Seq were then prepared with the TruSeq RNA Sample Prep Kits v2 (Illumina) following manufacturer instructions starting from the RNA fragmentation step.
The bioinformatic analysis of ChIP-Seq and RNA-Seq data was performed as described below.
RNA-Seq was performed on two biological replicates of four Eμ-myc Trp53KI/Δ lymphomas treated with 100 nM OHT or 1/1000 vol. ethanol for 2 h and three Eμ-myc Arf−/−Trp53+/+ lymphomas treated with 5 μ
The bioinformatic analysis of ChIP-Seq and RNA-Seq data was performed as described below.
4
Transcriptomic Analysis of B-cells
5
RNA Isolation, cDNA Synthesis, and RNA-Seq Library Preparation
6
RNA-Seq Library Preparation and Sequencing
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Quality and integrity of the total RNA was controlled on an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies; Waldbronn, Germany). The RNA sequencing library was generated from 100 ng total RNA using TruSeq RNA Sample Prep Kits v2 (Illumina, San Diego, CA, USA) for mRNA purification followed by ScriptSeq v2 RNA-Seq Library Preparation Kit (Epicentre, Illumina) according to manufacturer’s protocols. The libraries were sequenced on Illumina HiSeq2500 (Illumina), using TruSeq SBS Kit v3-HS (Illumina) (50 cycles, single ended run) with an average of 3 × 107 reads per RNA sample. Reads were aligned to the reference genome using open source short read aligner Tophat40 (link) followed by Cufflinks41 (link) that assembles transcripts, estimates their abundances, and tests for differential expression and regulation.
7
RNA Extraction and Sequencing Protocol
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Total RNA was extracted with a RNeasy Plant Kit (Qiagen, USA) following the manufacturer’s protocol. cDNA libraries for sequencing were constructed with the TruSeq RNA Sample Prep Kits v2 (Illumina) following the manufacturer’s protocol. An Illumina HiSeq2000 was used for sequencing with a 50 bp read length and a sequence depth of 20 million uniquely mapped reads.
8
RNA-seq Library Preparation and Sequencing
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As previously described (Rao et al., 2014 (link)), 1 μg of total RNA for each sample was used to construct an RNA sequencing (RNA-seq) library using TruSeq RNA Sample Prep Kits v2 (Illumina Inc., San Diego, CA, USA), according to the manufacturer’s instructions, at the Genomics Core Facility at the Noble Foundation (Ardmore, OK, USA). Each library was indexed. Six libraries with different indexes were pooled together in one lane for 100bp paired-end sequencing. The Hiseq2000 run was conducted at the Genomics Core Facility of the Oklahoma Medical Research Foundation (Oklahoma City, OK, USA).
9
Transcriptome Analysis of Arabidopsis Seedlings
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Total RNA was extracted from 9-day-old seedlings grown on 1/2MS medium horizontally. Illumina cDNA libraries were constructed with the TruSeq RNA Sample Prep Kits v2 (Illumina, San Diego, CA, USA) following the manufacturer’s protocol. Sequencing of the cDNA libraries was performed by pair-end methousing an Illumina HISEQ-x10 with a 150-bp read length and a sequence depth ~20 million uniquely mapped reads. Three biological replicates per sample. The data presented in the study are deposited in the NCBI repository, the BioProject ID is PRJNA971388.
10
Total RNA Extraction and Library Preparation
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Total RNA from tissues was purified using TRIzol (Invitrogen). Briefly, tissue was homogenised in TRIzol (Invitrogen) and chloroform was added. After centrifugation, RNA was purified using RNeasy Mini columns (Qiagen) according to the manufacturer’s protocol, including the on column DNaseI digestion step. Alternatively, total RNA was purified using PureLink RNA Mini Kit (Thermo Scientific) together with on column DNA digestion using PureLink DNase (Thermo Scientific). Then 1–5 µg of purified RNA was treated with Ribozero rRNA removal kit (Illumina) and ethanol precipitated. RNA quality and removal of rRNA were checked with the Agilent 2100 Bioanalyser (Agilent Technologies). Libraries for RNA-seq were then prepared with the TruSeq RNA Sample Prep Kits v2 (Illumina) or NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (NEB), following manufacturers instructions starting from the RNA fragmentation step.
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