Total RNA libraries were constructed using the Illumina TruSeq Stranded Total RNA Preparation Kit (Illumina) according to the manufacturer's guide. Input RNA was rRNA reduced using the Ribo-Zero Magnetic Kit (Human/Mouse/Rat), (Epicentre), and rRNA reduction was verified by BioAnalyzer 2100 (Agilent Technologies). Libraries were quality controlled and quantitated using the BioAnalzyer 2100 system and mass measurement using QuBit (Life Technologies). The libraries were clonally amplified on a cluster generation station using Illumina version 2 MiSeq reagents to achieve a target density of approximately 1000 K–1200 K clusters/mm2 on the flow cell. The resulting libraries were then sequenced on an Illumina MiSeq system at single reads of 50 bp, and were processed on the system to generate quality control metrics and FASTQ files with MiSeq Reporter 2.3.32.
Truseq stranded total rna preparation kit
Manufactured by Illumina
Sourced in United States
The TruSeq Stranded Total RNA Preparation Kit is a lab equipment product designed for the preparation of stranded total RNA samples. It enables the generation of strandspecific RNA sequencing libraries from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Hiseq 2500, by Illumina (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Allprep dna rna mirna universal kit, by Qiagen (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Kapa library quantification kit, by Roche (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Ribo zero magnetic kit human mouse rat, by Illumina (1 mentions)
Miseq system, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
2100 bioanalyser, by Agilent Technologies (1 mentions)
Nd 2000, by Thermo Fisher Scientific (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
Agilent d1000 screentape, by Agilent Technologies (1 mentions)
Direct zol rna miniprep kit, by Zymo Research (1 mentions)
Ribo zero gold, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
17 protocols using truseq stranded total rna preparation kit
1
Illumina TruSeq Stranded Total RNA Preparation
2
Transcriptome Analysis via RNA-seq
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The total RNA was extracted using a RNAprep pure Plant Kit (Tiangen, China) according to the manufacturer’s instructions, and the genomic DNA was removed using DNase I. The RNA quality and quantity were determined using a 2100 Bioanalyser (Agilent) and ND-2000 (NanoDrop Technologies) respectively. Then 5 μg of high-quality RNA (OD260/280 = 1.8~2.2, OD260/230 ≥ 2.0, RIN ≥ 6.5, 28 S:18 S ≥ 1.0) was used to construct the sequencing library.
The RNA-seq libraries were prepared according to the manufacturer’s instruction from the TruSeq Stranded Total RNA preparation Kit with Ribo-Zero Plant (Illumina, CA). cDNA synthesis, end repair, A-base addition, and ligation of the Illumina-indexed adaptors were performed according to Illumina’s protocol. Libraries were then size selected for cDNA target fragments of 200–300 bp on a 2% Low Range Ultra Agarose gel, followed by PCR amplification using Phusion DNA polymerase (NEB) for 15 cycles. After quantification by TBS380, the paired-end libraries were sequenced with 2 × 100-bp (PE100) reads on an Illumina HiSeq 2500 instrument.
The RNA-seq libraries were prepared according to the manufacturer’s instruction from the TruSeq Stranded Total RNA preparation Kit with Ribo-Zero Plant (Illumina, CA). cDNA synthesis, end repair, A-base addition, and ligation of the Illumina-indexed adaptors were performed according to Illumina’s protocol. Libraries were then size selected for cDNA target fragments of 200–300 bp on a 2% Low Range Ultra Agarose gel, followed by PCR amplification using Phusion DNA polymerase (NEB) for 15 cycles. After quantification by TBS380, the paired-end libraries were sequenced with 2 × 100-bp (PE100) reads on an Illumina HiSeq 2500 instrument.
3
Lung Transcriptomic Profiling in Pulmonary Hypertension
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After collecting lung tissue samples from normotensive and PH mice, total RNA was
isolated from lungs using an AllPrep DNA/RNA/miRNA Universal Kit (Qiagen) according to the
manufacturer’s instructions. RNA was quantified using NanoDrop™ (ThermoFisher), and
quality was assessed using an Agilent 2100 Bioanalyzer. RNA-seq library were constructed
using a TruSeq Stranded Total RNA Preparation kit (Illumina) with 200 ng of RNA as input
according to the manufacturer’s instructions. Libraries were sequenced on a HiSeq 2500
System (Illumina) with single-end reads of 100 nt at the University of Rochester Genomics
Research Center. Single-end sequencing was done at a depth of 10 million reads per
replicate (n=3). Quantitative analysis, including statistical analysis of differentially
expressed genes, was conducted with Cufflinks 2.0.2 and Cuffdiff2
(http://cufflinks.cbcb.umd.edu). The Benjamini-Hochberg method was applied for multiple
test correction (FDR < 0.05).
isolated from lungs using an AllPrep DNA/RNA/miRNA Universal Kit (Qiagen) according to the
manufacturer’s instructions. RNA was quantified using NanoDrop™ (ThermoFisher), and
quality was assessed using an Agilent 2100 Bioanalyzer. RNA-seq library were constructed
using a TruSeq Stranded Total RNA Preparation kit (Illumina) with 200 ng of RNA as input
according to the manufacturer’s instructions. Libraries were sequenced on a HiSeq 2500
System (Illumina) with single-end reads of 100 nt at the University of Rochester Genomics
Research Center. Single-end sequencing was done at a depth of 10 million reads per
replicate (n=3). Quantitative analysis, including statistical analysis of differentially
expressed genes, was conducted with Cufflinks 2.0.2 and Cuffdiff2
(http://cufflinks.cbcb.umd.edu). The Benjamini-Hochberg method was applied for multiple
test correction (FDR < 0.05).
4
YTHDF2 Depletion in MYC-ER HMECs
5
RNA-seq Analysis of Huh7 Cells and MSC Exosomes
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The mRNA expression pattern of Huh7 cells from the above four groups and MSC exosomes were analyzed by RNA-seq. RNA-seq libraries were prepared using the TruSeq Stranded Total RNA Preparation kit (Illumina, San Diego, CA, USA) followed by sequencing on the Illumina HiSeq 2000 platform at Genergy Biotechnology (Shanghai, China). Data processing, principal component analysis (PCA), heatmap, differentially expressed gene (DEG), volcano plot, and Venn diagram were analyzed using R-project (Version 3.6.3). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment in DEGs were performed by DAVID (https://david.ncifcrf.gov/ ). The corresponding upregulated DEGs in the comparison of MSC_Exo vs. Normal and MSC vs. MSC_GW and high expression gene of MSC exosomes (Top 500) were analyzed by the Venn diagram and ranked by the average log2FC.
6
Transcriptomic Analysis of P. xylostella
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Thirty 3rd instar P. xylostella larvae were collected in a PE tube as one sample. Trizol Reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA according to the manufacturer’s instructions. RNA degradation and contamination were assessed on 1% agarose gels and RNA concentration was measured using a NanoDrop 2000 (Thermo Fisher Scientific Inc, USA).
Library construction and RNA-Seq were performed by the OE Biotechnology Corporation (Shanghai, China). Total RNA from 9 samples (three independent biological replicates for each of the CHS, CHR and ZZ strains) with RNA integrity number (RIN) values above 8 were used to construct RNA-Seq libraries using the TruSeq stranded total RNA preparation kit with Ribo-Zero Gold (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Sequencing was performed on the Illumina HiSeq™2500 and 150 bp paired-end reads were generated.
Library construction and RNA-Seq were performed by the OE Biotechnology Corporation (Shanghai, China). Total RNA from 9 samples (three independent biological replicates for each of the CHS, CHR and ZZ strains) with RNA integrity number (RIN) values above 8 were used to construct RNA-Seq libraries using the TruSeq stranded total RNA preparation kit with Ribo-Zero Gold (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Sequencing was performed on the Illumina HiSeq™2500 and 150 bp paired-end reads were generated.
7
RNA-seq Library Preparation from Cell Pellets
8
Transcriptome profiling of SPIN1 knockdown and EML631 treated T778 cells
9
RNA-seq of LINC00355 Knockdown in MGC803
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Total RNA from control, LINC00355 knockdown (48 h after siRNA transfection) in MGC803 cells was isolated with RNeasy Mini Kit (Qiagen, Hilden, Germany). The experiment was performed with three independent biological replicates for each condition. The nine libraries were constructed with an Illumina TruSeq Stranded Total RNA Preparation Kit (Illumina, RS-122-2301), which contained Ribo-Zero Gold, which facilitates the depletion of rRNA. RNA-seq data are included in Supplementary Table 1 .
10
Whitefly Transcriptome Profiling Protocol
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Total RNA from each individual whitefly sample was used for cDNA library preparation using the Illumina TruSeq Stranded Total RNA Preparation kit as described by the manufacturer (Illumina, San Diego, CA, USA). Later on, sequencing of 10 samples was carried out using the HiSeq2000 on a rapid run mode generating 2x50 bp paired end reads. Base calling, quality assessment and image analysis were conducted using the HiSeq control software v1.4.8 and Real Time Analysis v1.18.61 at the Macrogen Korea.
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