Total RNA was extracted using the Maxwell® RSC simplyRNA Cells Kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. Library preparation was carried out with the TruSeq RNA Exome protocol (Illumina, San Diego, CA, USA) [50 (link)]. The synthesis of the cDNA was made using the SuperScript II Reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The cDNA adenylation and ligation at the 3′ end was made with adaptors. The library was amplified with PCR and clean-up took advantage of the AMPure XP beads (Beckman Coulter, Brea, CA, USA). The library was validated and the hybridization step was performed. To increase specificity against the regions that had to be captured, a second hybridization step was performed as before. The library was validated using the Tape Station. After the final step of normalization, the MiSeq Reagent Kit v3 by Illumina was used to sequence the library in single read mode on the MiSeq Instrument.
Truseq rna exome protocol
Manufactured by Illumina
Sourced in United States
The TruSeq RNA Exome protocol is a lab equipment product designed for targeted RNA sequencing. It enables the capture and enrichment of coding regions within the human transcriptome. The protocol provides a streamlined workflow for preparing RNA samples for sequencing on Illumina platforms.
Automatically generated - may contain errors
Lab products found in correlation
Maxwell rsc simplyrna cells kit, by Promega (4 mentions)
Bioanalyzer gel, by Agilent Technologies (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Labchip gx, by Agilent Technologies (2 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
7 protocols using truseq rna exome protocol
1
Comprehensive RNA Sequencing Workflow
2
RNA-seq Analysis of Macrophage Transcriptome
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We used a similar approach for RNA_Seq analysis as previously described23 (link). Three experimental conditions including M0 macrophages, control SC siRNA, and A3A siRNA (KD) are used. SC and KD samples were derived from the same three donors, whereas the M0 macrophages were derived from three unrelated donors. RNA libraries were prepared using the Illumina TruSeq RNA Exome protocol. For each sample, a total of ~90 million paired-end reads were obtained and QCed using fastqc (v0.10.1)49 . The reads were mapped to GRCh38 human reference genome and GENCODE (v25) annotation database using TopHat (v2.0.13)50 (link) with a maximum of 1 mismatch per read. The aligned reads were further checked with RSeQC (v2.6.3)51 (link) to identify potential RNASeq-related problems. Gene level read counts were estimated with featureCounts from Subread (v1.6.0)52 (link) using—fracOverlap 1 option. Differential expression analyses were performed using DESeq2 (v1.18.1)53 (link) and the heatmaps were generated using pheatmap (v1.0.8) R library54 . Pathways analyses were performed running GSEA (v3.0b3)55 (link) pre-ranked mode with genes ordered by DESeq2’s test statistic and MiSigDB canonical curated gene sets (c2).
3
RNA Extraction and RNA-seq Analysis
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In order to obtain RNA from the cell pellets, we followed the manufacturer’s instructions of the Maxwell® RSC simplyRNA Cells Kit (Promega, Madison, WI, USA). For the preparation of the library, TruSeq RNA Exome protocol (Illumina, San Diego, CA, USA) was used as already reported [39 (link)]. The library was analyzed using the Illumina instrument Miseq. The read quality was confirmed using fastqc (version 0.11.4, Babraham Institute, Cambridge, UK) and Trimmomatic (version 0.38, Usadel Lab, Aachen, Germany) [40 (link)] was used to remove poor quality reads and adapters. The cleaned reads were then aligned against the mouse reference genome GRCm39 and the annotation file version M28 using the Spliced Transcripts Alignment to a Reference (STAR, version 2.7.10a, New York, NY, USA) [41 (link)] and the final count was provided with htseq-count (version 0.6.1p1, European Molecular Biology Laboratory (EMBL, Heidelberg, Germany) [42 (link)]. Finally, the differentially expressed genes (DEGs) were obtained with the DESeq2 [43 (link)] Bioconductor [44 (link)] library of R (version 3.6.3, R Core Team).
4
CBG-Induced Transcriptome Analysis
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After 24 h of treatment with CBG at a concentration of 10µM, the cells were harvested and centrifugated, and the supernatant was discarded. The pellet was used for RNA extraction, along with the Maxwell® RSC simplyRNA Cells Kit (Promega, Madison, WI, USA), which was used followed the manufacturer’s instructions. TruSeq RNA Exome protocol (Illumina, San Diego, CA, USA) was used to perform library preparation, in compliance with the manufacturer’s instructions.
5
RNA Extraction and Exome Sequencing
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Maxwell® RSC simplyRNA Cells Kit (Promega, Madison, WI, USA) was used to extract DNA from cell pellets after harvesting, following the manufacturer’s instructions. As previously reported, the library was prepared using TruSeq RNA Exome protocol (Illumina, San Diego, CA, USA) [75 (link)].
6
RNA-seq Library Preparation Pipeline
7
RNA Sequencing Library Preparation
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Total RNA quality was determined by estimating the A260/A280 and A260/A230 ratios by nanodrop. RNA integrity was determined by running an Agilent Bioanalyzer gel, which measured the percentage of RNA fragments greater than 200nt for each sample (also called DV200). RNA Seq Library Prep: Following the Illumina TruSeq RNA Exome protocol (Cat. No. RS-301-2001), samples were binned based on DV200 scores. High quality samples had a DV200 > 70%, medium quality samples had a DV200 of 50-70%, low quality samples had a DV200 of 30-50%, and samples with a DV200 < 30% were not used. The RNA was fragmented using divalent cations under elevated temperature at various time periods based on DV200. cDNA was generated from the cleaved RNA fragments using random priming during first and second strand synthesis. Then, sequencing adapters were ligated to the resulting double-stranded cDNA fragments. The coding regions of the transcriptome were captured from this library using sequence-specific probes to create the final library. Indexed libraries that meet appropriate cut-offs for both were quantified by qRT-PCR using a commercially available kit (KAPA Biosystems) and insert size distribution determined with the Perkin Elmer LabChip GX or Agilent Bioanalyzer. Samples with a yield of ≥0.5 ng/ul were used for sequencing.
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