Truseq kit

Manufactured by Illumina

Sourced in United States, China, Hong Kong

The TruSeq kit is a library preparation kit designed to generate sequencing-ready libraries from DNA or RNA samples. The kit includes reagents and protocols for fragmentation, end-repair, adaptor ligation, and PCR amplification of nucleic acid samples prior to sequencing on Illumina platforms.

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Lab products found in correlation

Hiseq 2000, by Illumina (55 mentions) Hiseq 2500, by Illumina (30 mentions) Trizol reagent, by Thermo Fisher Scientific (20 mentions) Trizol, by Thermo Fisher Scientific (20 mentions) Hiseq platform, by Illumina (20 mentions) Rneasy mini kit, by Qiagen (17 mentions) Hiseq 2000 platform, by Illumina (15 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (14 mentions) Bioanalyzer, by Agilent Technologies (12 mentions) Hiseq 2000 sequencer, by Illumina (12 mentions) Nanodrop, by Thermo Fisher Scientific (12 mentions) Hiseq 4000, by Illumina (11 mentions) Dnaeasy genomic extraction kit, by Qiagen (11 mentions) Nextseq 500, by Illumina (9 mentions) 2100 bioanalyzer, by Agilent Technologies (8 mentions) Agilent bioanalyzer, by Agilent Technologies (8 mentions) High sensitivity dna chip, by Agilent Technologies (7 mentions) Hiseq 2000 system, by Illumina (6 mentions) Mrna seq sample prep kit, by Illumina (5 mentions) Rneasy plant mini kit, by Qiagen (5 mentions)

346 protocols using truseq kit

Estrogen Response in Breast Cancer Cells

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Human MCF-7 breast cancer cell line (ER positive) was cultured in Dulbecco's modi ed Eagle's medium (DMEM, Fisher, USA) given with 1% penicillin and 10% FBS under 37 °C in 5% CO 2 . E2-starved cells were conducted within 5% charcoal-stripped FBS in phenol-red-free DMEM for 72 h and treated with 10 nM 17-β-estradiol (E2). Control groups were only treated with the ethanol vehicle. Cells were collected after 48 h of experimental treatment and total RNA was harvested by using an RNAeasy Mini kit following the manufacturer's protocols (Qiagen, Valencia, CA). Two biological replicates of RNA-Seq and three biological replicates of Ribo-Seq were sampled both before and after the estrogen treatment.
Library preparation and high-throughput sequencing RNA-Seq stranded libraries were prepared with Illumina Truseq kit, with a paired-end sequencing strategy (150-nt). For Ribo-Seq, the stranded libraries were prepared with Illumina Truseq kit, with paired-end (40-nt), based on a previously described robust ribosome pro ling technique (Ingolia et al., 2009; Ingolia et al., 2011) . All samples were sequenced with Illumina NextSeq sequencer to acquire paired-end sequencing reads. All raw sequence reads can be accessible from the NCBI's Sequence Read Archive SRA database (BioProject ID: PRJNA639213).

Choo S.W., Zhong Y., Sendler E., Goustin A.S., Cai J., Ju D., Brown J., Kosir M.A., & Lipovich L. (2020). Integration of RNA-seq and Ribosome Profiling to Characterize Transcriptional and Translational Estrogen Responses of Genes in Human Breast Cancer.

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piRNA Identification from Oocyte Extracts

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XIWI and XILI were immunoprecipitated from stage 1–3 oocyte extract. Coprecipitating RNAs were purified with TRIzol and built into libraries using an Illumina Tru-seq kit without any further selection. Total RNA was purified from stage 1–3 oocyte extract with TRIzol and poly(A) RNA was purified with oligo(dT) beads and built into libraries using the Illumina Tru-seq kit. Coprecipitating piRNAs were separated on a 15% polyacrylamide TBE-Urea gel. Small piRNA sized bands were excised from gel and built into libraries based on the Ribosome Profiling library method starting at step 18 (Ingolia et al. 2012 (link)).

Toombs J.A., Sytnikova Y.A., Chirn G.W., Ang I., Lau N.C, & Blower M.D. (2017). Xenopus Piwi proteins interact with a broad proportion of the oocyte transcriptome. RNA, 23(4), 504-520.

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RNA-Seq Library Preparation and Analysis

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The quantity of total RNA in each sample was collected using Qubit (Invitrogen) and libraries prepared using Illumina TruSeq Total RNA v2 kit with Ribo Zero Gold for rRNA removal. The Ribo-Zero kit was used to remove rRNA (rRNA) from 1 μg of total RNA using a hybridization/bead capture procedure that selectively binds rRNA species using biotinylated capture probes. The resulting purified mRNA was used as input for the Illumina TruSeq kit in which libraries are tagged with unique adapter indices. Final libraries were validated using the Agilent high-sensitivity DNA kit (Agilent), quantified via Qubit, and diluted and denatured per Illumina’s standard protocol. High-throughput sequencing was carried out using the Illumina HiScan SQ instrument, 100 cycle paired-end runs, with 1 sample loaded per lane, yielding, on average, >100 million reads per sample. Reads were mapped to human genome hg19 using TopHat2 version 2.1.0^{17 (link)} with default settings and reads summarized by gene features using htseqcount. Differential expression analysis was performed and the p values adjusted for false discovery rates (FDR) were computed with DeSeq^{18 (link)} Data are available from GEO (accession no. GSE95670).

Ewing R.M., Song J., Gokulrangan G., Bai S., Bowler E.H., Bolton R., Skipp P., Wang Y, & Wang Z. (2018). Multiproteomic and Transcriptomic Analysis of Oncogenic β-Catenin Molecular Networks. Journal of proteome research, 17(6), 2216-2225.

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Sequencing small RNA from biofluids

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All sequencing data is available through the ERCC exRNA Atlas and through accession number phs001258.v1.p1 in dbGaP. The plasma, saliva and urine RNA were quantified in triplicate using Quant-iT Ribogreen RNA Assay kit, Low-Range protocol (R11490; ThermoFisher). The Illumina small RNA TruSeq kit (RS-200–0048; Illumina) was used for sequencing all samples. RNA input for plasma and saliva was 10–20 ng for all samples and the RNA input for urine was 30 ng for all samples. The reagents from the Illumina TruSeq kit were halved, as in Burgos et al., 2013. Each sample was assigned one of 48 possible indices. We used 16 PCR cycles for all samples. Indexed samples were run on a gel and purified away from the adaptor band. The samples were then pooled and placed on Illumina V3 single read flowcells (GD-401-3001; Illumina). The average read counts at each nucleotide length for each biofluid is displayed in Supplementary Figure S3.

Yeri A., Courtright A., Reiman R., Carlson E., Beecroft T., Janss A., Siniard A., Richholt R., Balak C., Rozowsky J., Kitchen R., Hutchins E., Winarta J., McCoy R., Anastasi M., Kim S., Huentelman M, & Van Keuren-Jensen K. (2017). Total Extracellular Small RNA Profiles from Plasma, Saliva, and Urine of Healthy Subjects. Scientific Reports, 7, 44061.

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Bru-Seq: Mapping Transcriptional Dynamics in PANC-1 Cells

Cited 3 times

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Bru-seq was performed as previously described (14 (link),16 (link)). Briefly, PANC-1 cells were incubated in bromouridine (Bru) (2 mM, Sigma-Aldrich) contained media for 30 minutes at 37°C. Cells were then lysed in Trizol, and total RNA was isolated. Bru-labeled RNA was immunocaptured using anti-BrdU antibodies (BD Pharmingen, 555627). Then strand-specific cDNA libraries were prepared using the Illumina TruSeq kit (Illumina), followed by deep sequencing using the Illumina sequencing platform as previously described (14 (link),17 (link),18 (link)).
The difference in expression profile was pre-ranked based on rLogFC value with a cut-off of 300 bp to eliminate the signal from noise or background. The pre-ranked file was loaded into the GSEA software tool (Broad Institute, Inc., Massachusetts Institute of Technology, and Regents of the University of California) for analysis of the upregulated pathways that are enriched in a positive or negative manner. The list of the top 10 gene sets is represented by a bar graph based on their Normalized enrichment score (NES).

Yang J., Jin L., Kim H.S., Tian F., Yi Z., Bedi K., Ljungman M., di Magliano M.P., Crawford H, & Shi J. (2022). KDM6A loss recruits tumor-associated neutrophils and promotes neutrophil extracellular trap formation in pancreatic cancer. Cancer research, 82(22), 4247-4260.

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Strand-Specific RNA Sequencing of Bromouridine-Labeled Transcripts

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bromouridine labeling, isolation of Bru-RNA and strand specific cDNA library preparation were performed as described previously (Paulsen et al., 2014 (link); Paulsen et al., 2013 (link)). Briefly, bromouridine (Aldrich) was added to the media of HeLa cell to a final concentration of 2 mM at 37° C for 30 min. Cells were then directly lysed by Trizol and total RNA isolated. The Bru-labeled RNA was captured from the total RNA by incubation with anti-BrdU antibodies (BD Biosciences) conjugated to magnetic beads under gentle agitation at room temperature for 1 h. Isolated Bru-labeled RNA was then used to prepare strand-specific DNA libraries using the Illumina Tru Seq Kit (Illumina) according to the manufacturer’s instructions with modifications previously described (Paulsen et al., 2014 (link)).
Reagents and other experimental procedures including immunoprecipitation, GST-pull down assays and RNA-seq, ChIP-seq and BrU-seq were described in Supplemental Information.

Zhang H., Gan H., Wang Z., Lee J.H., Zhou H., Ordog T., Wold M.S., Ljungman M, & Zhang Z. (2017). RPA Interacts with HIRA and Regulates H3.3 Deposition at Gene Regulatory Elements in Mammalian Cells. Molecular cell, 65(2), 272-284.

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Genome-wide DNA Methylation Profiling

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The genomic DNA was sheared using the Covaris sonicator and 100–300 bp fragments were obtained. DNA purification was conducted using AMPure beads. Illumina TruSeq kit was used to end repair the purified fragmented DNA and for ‘A’ tailing. TruSeq DNA adapters containing 5-methylcytosines were ligated to the sheared DNA and the ligated DNA was further purified using AMPure beads. Bisulfite treatment of the ligated DNA fragments was carried out using EZ DNA Methylation-Gold Kit (Zymo Research) following the manual’s instructions. The bisulfite treated DNA fragments were PCR amplified using the PCR primer cocktail (Illumina TruSeq kit). The bisulfite libraries, thus constructed, were purified using AMPure beads and subjected to quality testing using Bioanalyzer (Agilent 2100). The library (~10 pM) was sequenced using the Illumina HiSeq 2500 machine for 90 cycles in the paired-end mode.

Verma P., Singh A., Purru S., Bhat K.V, & Lakhanpaul S. (2022). Comparative DNA Methylome of Phytoplasma Associated Retrograde Metamorphosis in Sesame (Sesamum indicum L.). Biology, 11(7), 954.

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Erythroblast RNA-seq Analysis Protocol

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RNA was extracted from ES‐derived Day 17 erythroblasts which are orthochromatic erythroblasts using QIAGEN RNA isolation kit (QIAGEN#74104) according to the manufacturer's instructions. The RNA integrity number of each sample was >9. Approximately 100 ng of total RNA was used for construction of cDNA library by Illumina TruSeq kit (Illumina #RS‐121‐2001, #RS‐121‐2002). Sequencing was performed on Illumina HiSeq 4000 with 150 bp paired‐end. RNA‐Seq data of CB‐derived erythroblasts were downloaded from our previous data.³³ Raw bulk RNA‐seq data of ES‐ortho were filtered by fastp to remove low quality reads. Reads were mapped to UCSC hg19 by STAR³⁴ and quantified by FeatureCounts.³⁵ Gene expression was analysed using DESeq2.³⁶ Principal component analysis (PCA) was performed on log‐transformed normalized counts for expressed genes. Differentially expressed genes were identified as fold change ≥4 and adjusted p‐value ≤0.05. GO (Gene ontology) enrichment analysis were performed by Metascape.³⁷ GO term was considered as significant with q‐value <0.001. RNA‐seq data are available at GEO under accession number GSE179778. Time series analysis was applied by R package TCseq.

Wang S., Zhao H., Zhang H., Gao C., Guo X., Chen L., Lobo C., Yazdanbakhsh K., Zhang S, & An X. (2022). Analyses of erythropoiesis from embryonic stem cell‐CD34+ and cord blood‐CD34+ cells reveal mechanisms for defective expansion and enucleation of embryomic stem cell‐erythroid cells. Journal of Cellular and Molecular Medicine, 26(8), 2404-2416.

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RNA Extraction and Small RNA Sequencing

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Soybean seeds stored at −80 °C were ground under liquid nitrogen conditions. Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA, Cat no.15596-026) for RNA-seq and small RNA-seq [13 (link)]. A NanoDrop instrument was used to determine RNA integrity and purity and an OD value between 1.8 and 2.2 was required. Electrophoresis detection of 28S:18S was at least 1.5, and the total RNA concentration was not less than 750 ng/μg. Small RNA was isolated and purified from total RNA, and small RNAs of 18-30 nt in length were selected. Using the Small RNA Sample Prep Kit, 3′ and 5′ linkers were connected to the samples that met the qualifications, and subsequently subjected to reverse transcription amplification. Libraries were constructed using the Illumina TruSeq kit (RS-200-0012; Illumina Inc., San Diego, CA, USA). Then, Reverse Transcription PCR (RT-PCR), purification of the small RNA library, library detection (electrophoresis detection, NanoDrop and Agilent Technologies 2100 analyzer) and PCR amplification were performed in sequence. The target-sized fragment was separated by PAGE gel electrophoresis. Finally, high-throughput sequencing was performed on the Illumina platform (Illumina HiSeq 4000 SE 150).

Yu J.Y., Zhang Z.G., Huang S.Y., Han X., Wang X.Y., Pan W.J., Qin H.T., Qi H.D., Yin Z.G., Qu K.X., Zhang Z.X., Liu S.S., Jiang H.W., Liu C.Y., Hu Z.B., Wu X.X., Chen Q.S., Xin D.W, & Qi Z.M. (2019). Analysis of miRNAs Targeted Storage Regulatory Genes during Soybean Seed Development Based on Transcriptome Sequencing. Genes, 10(6), 408.

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BrU-Seq and BruChase-Seq of T cell RNA

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Bromouridine (BrU, Aldrich, cat# 850187) was added to the culture media of 10 million resting or activated CD4⁺ T cells to a final concentration of 2 mM. After incubation at 37°C for 1 h, the cells were washed three times with PBS and either collected directly (nascent RNA, Bru-Seq) or chased in the conditioned cell-culture media containing 20 mM uridine for 0.5 h or 2 h at 37°C (0.5 h or 2 h RNA, BruChase-Seq) (24 (link),27 (link)). Total RNA was prepared using TRIzol Reagent (Invitrogen), and cytoplasmic RNA was isolated as described in (31 (link)). BrU labeled RNA was isolated from the total RNA or cytoplasmic RNA by anti-BrdU antibodies (BD Biosciences, cat# 555627) or mouse IgG (BD Biosciences, cat# 555746) conjugated to Dynabeads Goat anti-mouse IgG (Invitrogen, cat# 110.33) (24 (link),27 (link)). The isolated BrU-labeled RNA was used for constructing strand-specific RNA-Seq library with the Illumina TruSeq Kit (Illumina) according to the manufacturer's instructions. Raw sequencing data were acquired with an Illumina HiSeq-3000 at the DNA Sequencing and Genomic Core, NHLBI, NIH.

Tian Y., Zeng Z., Li X., Wang Y., Chen R., Mattijssen S., Gaidamakov S., Wu Y., Maraia R.J., Peng W, & Zhu J. (2020). Transcriptome-wide stability analysis uncovers LARP4-mediated NFκB1 mRNA stabilization during T cell activation. Nucleic Acids Research, 48(15), 8724-8739.

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