RNA samples for each shRNA construct (ETV4a, ETV4b, MED25a, MED25b, lenti-luciferase shRNA) were analyzed as biological triplicates except for ETV4 shRNA retroviral control, which had two biological replicates. RNA was prepared from cells at day 7 post-infection using the Qiagen RNAeasy Mini Kit. Samples were treated on column with DNase to remove genomic DNA. Samples were depleted of ribosomal DNA using a bead-based method (Illumina TruSeq Stranded RNA Kit with Ribo-Zero Gold) prior to library construction. Library construction was performed using the Illumina TruSeq Stranded RNA Kit and sequenced using the Illumina Hiseq2000 sequencer. Sequence reads were aligned to hg19 using novoalign (Novocraft) and differentially expressed transcripts determined with drds analysis package in Useq. Expression changes of an absolute log 2 fold change > 0.585 and FDR <1%, relative to control shRNA, were counted as significant. In order to be considered affected by both ETV4 and MED25 shRNA, the expression of a gene had to be significantly affected by all four shRNA constructs (ETV4a, ETV4b, MED25a, MED25b).
Truseq stranded rna kit
Manufactured by Illumina
Sourced in United States
The TruSeq Stranded RNA kit is a library preparation kit for RNA sequencing. It is designed to generate stranded RNA-Seq libraries from total RNA samples. The kit provides a streamlined workflow for library preparation, including steps for RNA purification, fragmentation, cDNA synthesis, and adaptor ligation.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 4000, by Illumina (7 mentions)
Hiseq 2500, by Illumina (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Hiseq 2500 platform, by Illumina (5 mentions)
Rneasy mini kit, by Qiagen (5 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Mirneasy kit, by Qiagen (3 mentions)
Qiazol lysis reagent, by Qiagen (3 mentions)
Ribo zero gold, by Illumina (3 mentions)
Novaseq 6000, by Illumina (3 mentions)
Novaseq, by Illumina (3 mentions)
Ribozero depletion gold kit, by Illumina (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Hiseq 2000 system, by Illumina (2 mentions)
Hiseq 2000 sequencer, by Illumina (2 mentions)
Truseq small rna kit, by Illumina (2 mentions)
Magnetic mrna isolation kit, by New England Biolabs (2 mentions)
Nextflex rapid directional rna seq kit, by PSC Biotech (2 mentions)
60 protocols using truseq stranded rna kit
1
Differential Transcriptomic Analysis of ETV4 and MED25 Knockdown
2
RNA Sequencing and Differential Gene Expression
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RNA was collected from T1 and T2 cell cultures polarized for 5 days using TRIreagent and complementary DNA synthesized using SuperScript First Strand Synthesis (ThermoFisher). Quantitative real time PCR measured expressed RNAs using SYBR green master mix (Applied Biosystems). RNA levels were standardized relative to the housekeeping gene GAPDH. Patient samples containing definite outliers (ROUT with Q=0.1% by GraphPad Prism software) were removed. Patient samples with both T1 and T2 available were included for analysis.
For RNA sequencing, RNA was collected by TRIreagent followed by DNase digestion. Library preparation was performed with the Illumina Tru-Seq Stranded RNA kit. RNA sequencing was performed with an Illumina HiSeq2500 instrument by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core facilities consecutively on all samples. 100bp paired end reads were generated. Average sequencing depth of all samples was 43 million mapped reads ± 13.5 million (standard deviation). FASTQ files were processed using DESeq2 to identify differentially expressed genes (33 (link)). Data are available in NCBI’s Gene Expression Omnibus (34 (link)) with GEO series accession number GSE185193. GO Enrichment analysis for overrepresented biological processes was performed using the Gene Ontogeny Resource (35 (link)–37 (link)).
For RNA sequencing, RNA was collected by TRIreagent followed by DNase digestion. Library preparation was performed with the Illumina Tru-Seq Stranded RNA kit. RNA sequencing was performed with an Illumina HiSeq2500 instrument by the Vanderbilt Technologies for Advanced Genomics (VANTAGE) core facilities consecutively on all samples. 100bp paired end reads were generated. Average sequencing depth of all samples was 43 million mapped reads ± 13.5 million (standard deviation). FASTQ files were processed using DESeq2 to identify differentially expressed genes (33 (link)). Data are available in NCBI’s Gene Expression Omnibus (34 (link)) with GEO series accession number GSE185193. GO Enrichment analysis for overrepresented biological processes was performed using the Gene Ontogeny Resource (35 (link)–37 (link)).
3
Transcriptome Sequencing Using Illumina HiSeq
4
Total RNA Extraction and Sequencing
5
Transcriptomic Analysis of Brain Calcifications
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Mice were deeply anesthetized and transcardially perfused with ice-cold PBS. Brains were removed, placed in RNAlater Stabilization Solution (Thermo Fisher Scientific, catalog no. AM7020), and 1-mm coronal sections were cut using a brain matrix (RBMA-200C, World Precision Instruments). Calcifications were detected on the basis of their autofluorescence using a fluorescent stereomicroscope (Zeiss Axio Zoom.V16) and were surgically removed together with surrounding tissue. Cortical sections were also removed as examples of non–calcification-prone regions. RNA was isolated with a micro RNA kit (Qiagen) according to the manufacturer’s instructions. The concentration of RNA and sample purity were assessed using a 2100 Bioanalyzer (Agilent) and RNA 6000 Pico Kit (Agilent). RNA samples were poly(A)-enriched, and libraries were prepared using the Illumina TruSeq Stranded RNA kit. RNA was sequenced on an Illumina platform HiSeq 4000 at the Functional Genomic Center Zurich (UZH, ETH). The Illumina single-read approach (1 × 125 base pair) was used to generate raw sequencing reads with a depth of 20 million to 30 million reads per sample.
6
RNA-Seq Library Preparation with rRNA Depletion
7
Transcriptome Profiling of Softwood Trees
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Tissues from the corresponding regions of SW-1, SW-2, and TZ of the frozen cross-section wood disks from 3 trees were used to isolate the total RNA according to a CTAB method [54 (link)]. Each of the frozen tissues were ground into fine powders in a freezer mill (IKA, A11) under liquid N2. The extraction buffer consisted of 2% CTAB, 2% PVP-40, 0.1 M Tris pH8.0, 25 mM EDTA, 2M NaCl, and 2% mercaptoethanol. RNA concentration and purity (A260/A280 and A260/A230) were measured with microvolume spectrophotometry (Nanodrop 2000, Thermo Scientific). A bioanalyzer (Agilent Technology) was used to evaluate the RNA qualities. Sequencing libraries were constructed by the Illumina TruSeq stranded RNA kit. Sequencing was performed by an Illumina NextSeq 500 sequencer provided by Genomics BioSci & Tech. (Taipei, Taiwan) to generate 150 bp paired-end reads. A total of 9 RNA-seq libraries, with 3 tissues (SW-1, SW-2, and TZ) per tree for 3 trees (Tree A, B, and C), were sequenced. The sequence data for these transcriptomes have been transferred to the National Center for Biotechnology Information (NCBI) under the SRA accession: PRJNA589955.
8
Illumina TruSeq Stranded RNA Sequencing
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Biological duplicates of sequencing libraries were prepared from high quality RNA extracts (50 ng exRNA and 500 ng cellular RNA) using the Illumina TruSeq Stranded RNA Kit according to the manufacturer’s instructions. The TruSeq PE Clusterkit v3-cBot-HS was used on an Illumina HiSEq 2000 machine.
9
Characterizing PDX Mammary Tumor Transcriptomes
10
RNA-Seq Analysis of Murine T Cells
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Brains were harvested and immune cells were isolated as described above. Ten mice per group were used for cell isolation. CD4+ and CD8+ T cells were enriched using the negative selection Easy Sep Mouse CD4+ T cell Isolation Kit and Easy Sep Mouse CD8+ T cell Isolation Kit (STEMCELL Technologies, Vancouver, Canada). RNA was immediately isolated using RNeasy (QIAGEN), according to the manufacturer’s protocol, and stored at -80°C. Illumina TruSeq stranded RNA Kit with Ribo-Zero Gold (Illumina, San Diego CA) was used to prepare cDNA libraries for RNA-Seq according to the manufacturer’s protocol. HiSeq 50 cycle single-read sequencing was performed on an Illumina HiSeq 2500 instrument. RNA-Seq and the bioinformatic analyses of the results obtained from the RNA-Seq experiment were done by the High Throughput Genomics Core Facility in the Huntsman Cancer Institute at the University of Utah.
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