RNA sequencing was performed by the Nucleomics Core Facility (VIB, Leuven, Belgium) on iPSC‐derived motor neurons. RNA was isolated using an RNeasy kit (Qiagen). From extracted RNA, libraries were made using the Illumina TruSeq Stranded mRNA Library protocol. These libraries were sequenced on an Illumina NextSeq 500 paired‐end 75 bp and yield an average of 90 million reads per sample. To estimate the expression of the transcript of every sample, reads were counted using Salmon (v0.8.1) (Patro et al, 2017 ) against the ensemble transcript for the human reference genome hg38. Gene expression from the protein‐coding transcripts was then estimated using the tximport function the R‐package tximport (v1.6.0) (Soneson et al, 2016 ). Bulk RNA‐sequencing data of iPSCs and astrocytes were obtained from GSE98290 and processed in the same way (Hall et al, 2017 ).
Truseq stranded mrna library protocol
Manufactured by Illumina
Sourced in United States
The TruSeq Stranded mRNA Library protocol is a library preparation method designed for RNA sequencing applications. It enables the generation of stranded mRNA libraries from total RNA samples. The protocol preserves the information about the original RNA strand orientation, which is important for certain downstream analyses.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy kit, by Qiagen (3 mentions)
Nextseq 500, by Illumina (2 mentions)
Novaseq s1 200, by Illumina (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Nextseq, by Illumina (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Hiseq 2500 sequencer, by Illumina (1 mentions)
Truseq dna protocol, by Illumina (1 mentions)
Direct zol rna extraction kit, by Zymo Research (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
9 protocols using truseq stranded mrna library protocol
1
RNA-seq of iPSC-derived motor neurons
2
Sea Lamprey Tissue Transcriptome Profiling
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In total, 63 sea lamprey tissue samples from six organs (brain, heart, liver, kidney, ovary and testis) were dissected from larval, juvenile and adult specimens. Total RNA was extracted using different extraction protocols (Supplementary Table 1 ); RNA quality was inspected using the Fragment Analyzer (Advanced Analytical Technologies), and its concentration was determined using a NanoDrop (Thermo Fisher Scientific). Strand-specific RNA-seq libraries were generated using the Illumina TruSeq Stranded mRNA Library protocol. Each library was sequenced on Illumina HiSeq 2500 platforms (100 nucleotides, single-end) at the Lausanne Genomic Technologies Facility (https://www.unil.ch/gtf ).
3
Sequencing E. heatwolei Genome and Transcriptome
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One adult male (Euhea_18_05) and one adult female individual (Euhea_18_03) of E. heatwolei species were captured from a population that inhabits Woods Reserve, Corin Road, ACT, Australia (−35.480751, 148.940398). Both individuals were sacrificed by intraperitoneal injection of pentobarbitone following the standard operating procedures specified by the animal ethics committee of the University of Canberra. We generated DNA-seq libraries for a male and female E. heatwolei from liver tissue using the Illumina TruSeq DNA protocol for short insert size (400–450 nt). We generated strand-specific RNA-seq libraries (using the Illumina TruSeq Stranded mRNA Library protocol) for a total of six samples obtained from brain, liver, and gonads for a male and female E. heatwolei. All libraries were sequenced on Illumina HiSeq 2500 sequencers at the University of Canberra. We generated 262–269 million 150-nt paired-end DNAseq reads. We generated 82–95 million 125-nt paired-end RNAseq reads. Further details in supplementary table 4 , Supplementary Material online . Quality of the reads was verified using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc , last accessed May 25, 2020) and the remaining adaptors were removed with Trimmomatic (Bolger et al. 2014 (link)).
4
Bacterial RNA Extraction and Sequencing
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Cell pellets were resuspended in trizol and stored at –80°C immediately after centrifugation and removal of supernatant. Upon extraction, pellets were thawed on ice. Total RNA was extracted using the Direct-zol RNA extraction kit from Zymo, San Diego, California. We used Ribo-Zero specific for bacteria to remove ribosomal RNA (Illumina, San Diego, California). Libraries were built using the Illumina TruSeq Stranded mRNA library protocol. Libraries were sent to the UC Irvine sequencing core for 250 paired end sequencing on the HiSeq 2500.
5
Zebrafish RNA-seq transcriptome analysis
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RNA-sequencing was performed by the Nucleomics Core Facility (VIB, Leuven, Belgium). RNA was isolated using the RNeasy kit (Qiagen). From extracted RNA, libraries were made using the Illumina TruSeq Stranded mRNA Library protocol. These libraries were sequenced on an Illumina NextSeq 500 paired-end 75 bp and yield an average of 37.5 million reads per sample (range 35.1–40.5). To estimate the expression of the transcript of every sample, reads were counted using Salmon (v0.14.0) [55 (link)] against ensemble transcript for the zebrafish reference genome GRCz11. Transcript level expression from the protein coding genes was summarized to the gene level expression using the R-package tximport (v1.12.0). Differential expression was performed with edgeR (v3.24.3) [43 (link)]. Genes with a FDR-adjusted P value smaller than 0.05 and with an altered expression of 30% were deemed significantly differentially expressed. Datasets are deposited and available in ENA (project accession PRJEB50765).
6
RNA-seq Analysis of Liver Samples
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RNA-seq libraries were prepared by IMB Sequencing Facility, University of Queensland, Australia, with the TruSeq Stranded mRNA Library protocol (Illumina, San Diego, California, USA). Sequencing of 12 liver samples (with 84 unrelated samples) was performed using a single NovaSeq S1 200 cycle sequencing run on a NovaSeq 6000 machine (Illumina) through IMB Sequencing Facility. Sequencing depth was between 9 million and 35 million paired end reads per sample. The raw sequencing data, in the form of.fastq files, are deposited in the European Nucleotide Archive under study accession number PRJEB39130.
7
RNA-seq Protocol for Transcriptome Analysis
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RNA sequencing was performed by the Nucleomics Core Facility (VIB, Leuven, Belgium). RNA was isolated using an RNeasy kit (Qiagen). From extracted RNA, libraries were made using the Illumina TruSeq Stranded mRNA Library protocol. These libraries were sequenced on an Illumina NextSeq 500 paired-end 75 bp and yield an average of 90.2 million reads per sample (range 75.6–106.9). To estimate the expression of the transcript of every sample, reads were counted using Salmon (v0.8.1)68 (link) against the ensembl transcript for the human reference genome hg38. Gene expression from the protein coding transcripts was then estimated using the tximport function the R-package tximport (v1.6.0)69 (link).
8
Transcriptome Analysis of Urosaurus Lizards
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Two males and two females of U. nigricaudus were captured in Santa Rita (23.3279699° N, −109.7016934° W) Baja California, Mexico. Tails were collected and flash-frozen in liquid nitrogen. Tissues were shipped to Cantata Bio LLC for total RNA extraction and library preparation. Additionally, one male and one female U. bicarinatus were captured near El Limón (18.531466° N, −98.9416673° W) in Morelos, Mexico. The specimens were housed for 1 day prior to being euthanized using ∼100 mg/km sodium pentobarbital solution via intracoelomic injection. Brain, heart, kidney, liver, and the gonads were collected and flash-frozen in liquid nitrogen and then stored at −80 °C. RNA was extracted, used to generate strand-specific RNA-seq libraries (using the Illumina TruSeq Stranded mRNA Library protocol), and sequenced on an Illumina NextSeq 500 platform at UNAM's sequencing facility (http://www.uusmb.unam.mx/ ).
9
Illumina NovaSeq-based RNA-seq Protocol
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RNA-seq libraries were prepared by IMB Sequencing Facility, University of Queensland, Australia, with the TruSeq Stranded mRNA Library protocol (Illumina, San Diego, California, USA). Sequencing was performed using a single NovaSeq S1 200 cycle sequencing run on a NovaSeq 6000 machine (Illumina) through IMB Sequencing Facility. Sequencing depth was between 9 million and 35 million paired end reads per sample. The raw sequencing data, in the form of .fastq files, have been deposited in the European Nucleotide Archive under study accession number PRJEB39130.
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