RNA was extracted from FFPE blocks using the truXTRAC FFPE total NA Ultra kit (Covaris). RNA quality with DV200 was examined using the 4200 TapeStation (Agilent Technologies), and RNA concentration was determined using the Qubit Fluorometric Quantitation (Thermo Fisher). Ribosome RNA was depleted using the QIAseq FastSelect (Qiagen), and stranded cDNA library for RNAseq was prepared using the TruSeq Stranded LT mRNA kit (Illumina), following the manufacturer’s protocols. Pooled libraries were paired-end sequenced (100 bp × 2) on the NextSeq 2000 system (Illumina). Raw sequence reads were de-multiplexed by the on-instrument DRAGEN (v3.8.4). Sequence reads of each sample were pseudo-aligned to the human hg38 reference transcriptome, and the gene transcript abundance was quantified using the Kallisto (v0.48.0). Differential gene expression was achieved using biomaRt (v2.50.3), tximport (v1.22.0) and DESeq2 (v1.34.0) packages in the R Studio (Build 386 with R v4.1.1).
Truseq stranded mrna lt kit
Manufactured by Illumina
Sourced in United States
The TruSeq Stranded mRNA LT Kit is a library preparation kit designed for RNA sequencing. The kit enables the preparation of stranded mRNA libraries from total RNA samples. It includes reagents and protocols for mRNA isolation, fragmentation, cDNA synthesis, adaptor ligation, and library amplification.
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Lab products found in correlation
Hiseq 3000 4000 sbs kit, by Illumina (14 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (9 mentions)
Rneasy mini kit, by Qiagen (8 mentions)
Trizol reagent, by Thermo Fisher Scientific (8 mentions)
Agilent bioanalyzer, by Agilent Technologies (8 mentions)
Hiseq 2500, by Illumina (7 mentions)
Nextseq 500, by Illumina (7 mentions)
Rna screentape, by Agilent Technologies (4 mentions)
Hiseq 2000, by Illumina (3 mentions)
Rneasy kit, by Qiagen (3 mentions)
Nextseq 500 sequencer, by Illumina (3 mentions)
Truseq sbs kit v4, by Illumina (3 mentions)
Tapestation 2200, by Agilent Technologies (3 mentions)
High output 75 cycles kit, by Illumina (3 mentions)
Qiacube connect, by Qiagen (3 mentions)
Tapestation 4200, by Agilent Technologies (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Nextseq 500 system, by Illumina (2 mentions)
Rneasy lipid tissue mini kit, by Qiagen (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
62 protocols using truseq stranded mrna lt kit
1
FFPE RNA Extraction and Transcriptomic Analysis
2
Hypothalamic Transcriptome Sequencing
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Total RNA was isolated from hypothalamic tissues as previously described. RNA quality was examined using the 4200 TapeStation (Agilent Technologies) and its concentration was determined with the Qubit Fluorometer (Thermo Fisher). Stranded cDNA libraries were prepared using the TruSeq Stranded LT mRNA kit (Illumina) in accordance with the manufacturer’s protocol using the poly-adenylated RNA isolation workflow. Sequencing of paired-end reads (100 bp × 2) was performed on a NextSeq 2000 platform (Illumina) at the Brody Integrative Genomics Core. Raw sequence reads were obtained from on-instrument DRAGEN (v3.8.0). Sequence reads of each sample were pseudo-aligned to the mouse reference (mm10) and the gene transcript abundance was quantified using Kallisto (v0.48.0). Differential expressed genes between experimental conditions were identified using DESeq2 (v1.34.0) in RStudio (Build 386 with R v4.4.1). P values lower than 0.05 after false-discovery rate (FDR) correction were considered significant.
3
RNA-Seq Library Preparation and Sequencing
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Total RNA was extracted as described in the RT-qPCR protocol section. Banks for deep-sequencing were prepared from 800 ng total RNA using Illumina's TruSeq Stranded mRNA LT Kit according to manufacturer's protocol. Samples were sequenced paired-end at 50-bp read length on an Illumina HiSeq-2000. As described in supplementary methods , the reads were trimmed using Trimmomatic, aligned onto the hg19 reference genome using TopHat2, filtered using SamTools, then the FPKM calculated using Cufflinks (21 –24 (link)).
4
mRNA Library Preparation and Sequencing
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The mRNA libraries were prepared using a TruSeq® Stranded mRNA LT kit (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. Sequencing was performed on the NextSeq500 System (read length—75 nt, single-end mode) using the NextSeq 500/550 High Output Kit v2.5 (Illumina).
5
Fetal Tissue Transcriptome Analysis
6
Transcriptome Profiling of HCT116 Cells
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Total RNA was extracted from HCT116 p53+/+ cells using GenElute (Sigma) and treated with DNaseI (Ambion). The libraries for deep-sequencing were prepared from 800 ng total RNA using Illumina’s TruSeq Stranded mRNA LT Kit according to manufacturer’s protocol. Samples were sequenced paired-end at 50-bp read length on an Illumina HiSeq-2000. See supplementary S1 File for biofinformatic procedures.
7
RNA-Seq Transcriptome Analysis Pipeline
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RNA was extracted using RNeasy Micro Kit (Qiagen), and libraries were prepared using TruSeq Stranded mRNA LT Kit (Illumina). Paired-end sequencing was performed using HiSeq 2500 (Illumina). Cutadapt[27 ] and FASTX-Toolkit (http://hannonlab.cshl.edu/fastx-toolkit ) were used to remove adaptor sequences and ends with phred quality scores less than 20. UCSC hg19 reference sequence (http://genome.ucsc.edu/ ) was used as the reference genome, and STAR[28 (link)] was used for mapping. Read count was obtained for each gene using HTSeq[29 (link)]. Read count after quality control was 4.9x106 ~ 9.7x106. Differential gene analysis was performed using edgeR 3.12.0[30 (link)]. Pathway analysis was performed by uploading genes with false discovery rate less than 0.05 by the Benjamini-Hochberg method and their logFCs into IPA software (Qiagen)[31 (link)]. R version 3.2.3 was used for RNA-Seq analysis.
8
B cell transcriptomic analysis upon YY1 deletion
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Mu-IgM/anti-CD40 stimulated follicular B cells were isolated from 3 month old female or male yy1flox/flox animals (three separate animals each) treated with or without TAT-CRE. About 48 hours post-stimulation and YY1 deletion, total RNA was isolated using TRIzol (Invitrogen) and libraries were prepared with an Illumina TruSeq Stranded mRNA LT kit. Samples were run on Illumina NextSeq 500 and bioinformatic analyses were conducted as previously described [75 (link)]. Briefly, R (v3.3.1), RStudio (v1.0.44), and the Bioconductor suite of packages (http://www.bioconductor.org ) were used to perform X-linked gene expression analyses. RNA-Seq reads were aligned with Tophat to the GRCm38/mm10 mouse reference genome and HTSeq-count and edgeR were used to normalize data. Statistical significance was determined with log2 fold change (lfc) and false discovery rates (FDR) (FDR <0.05, lfc > 0.5 or <-0.5). Heatmaps were constructed in RStudio (gplots, RColorBrewer), and expression levels for differentially expressed X-linked genes are shown. Gene Ontology (GO) term enrichment was performed using DAVID v6.8. A selection of Functional Annotation Clustering terms with an enrichment score >1 (p < 0.05) are shown. The RNAseq data are available in the Gene Expression Omnibus (GEO) database under the accession number GSE104097.
9
Striatum RNA-seq from F2 Mice
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Striatum punches were collected as described30 (link) for RNA-seq from 23 F2 mice (all OXY-treated for historical reasons). Brains were rapidly removed and sectioned with a brain matrix to obtain a 3-mm-thick section where a 2 mm diameter punch of the striatum was collected. Left and right striatum punches were pooled and immediately placed in RNAlater (Life Technologies, Grand Island, NY, USA) for 48 h prior to storage in a −80°C freezer. Total RNA was extracted using the RNeasy kit (Qiagen, Valencia, CA, USA) as described.30 (link) RNA was shipped to the University of Chicago Genomics Core Facility for cDNA library preparation using the Illumina TruSeq (oligo-dT; 100 bp paired-end reads). Libraries were prepared according to Illumina’s detailed instructions accompanying the TruSeq® Stranded mRNA LT Kit (Part# RS-122–2101). The purified cDNA was captured on an Illumina flow cell for cluster generation and sample libraries were sequenced at 23 samples per lane over five lanes (technical replicates) according to the manufacturer’s protocols on the Illumina HiSeq4000 machine, yielding an average of 69.4 million reads per sample. FASTQ files were quality checked via FASTQC and possessed Phred quality scores > 30 (i.e., less than 0.1% sequencing error).
10
Streamlining RNAseq: Optimizing Library Preparation and Downstream Analysis
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For RNAseq, RNA libraries were prepared from 100ng of total RNA per sample for 6 DO mice, 3 brain regions per mouse using the TruSeq stranded mRNA LT kit (Cat# RS-122–2101, Illumina). These synthetic RNAs cover a range of concentrations, length, and GC content for validation of the fidelity and dose-response of the library prior to downstream procedures. Libraries prepared with unique barcodes were pooled at equal molar ratios following manufacturer’s protocol (Cat# 15035786 v02, Illumina). The pool was denatured and subject to paired-end 50x sequencing on the Hi-Seq 2500 platform. An average of 67 million reads per sample were obtained. Sequencing reads were aligned to the mouse genome (mm10) using STAR (v2.4.2a) and aligned reads were quantified using Salmon (v0.8.2). Approximately 90% of the reads mapped uniquely. Hierarchical clustering and Principal Components Analysis were performed following Variance Stabilizing Transformation (VST) from DESeq2, which is on the log2 scale and accounts for library size differences. The hierarchical clustering heatmap shows the Euclidean distances of VST of the counts between samples.
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