Truseq rna sample prep v2 ls protocol

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The TruSeq RNA sample Prep v2 LS protocol is a laboratory equipment product designed for the preparation of RNA samples for sequencing. It provides a standardized workflow to convert RNA into a library of template molecules suitable for subsequent cluster generation and sequencing. The protocol includes steps for RNA fragmentation, cDNA synthesis, adapter ligation, and library amplification.

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Lab products found in correlation

Mirneasy kit, by Qiagen (2 mentions) Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions) Nanodrop 2000, by Thermo Fisher Scientific (2 mentions) Hiseq 2500 flow cell, by Illumina (1 mentions) Quick amp labeling kit, by Agilent Technologies (1 mentions) Feature extraction software, by Agilent Technologies (1 mentions) Gaiix platform, by Illumina (1 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions) Gaiix, by Illumina (1 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions) Ez link hpdp biotin, by Thermo Fisher Scientific (1 mentions) μmacs streptavidin microbeads, by Miltenyi Biotec (1 mentions) Minelute spin column, by Qiagen (1 mentions) Hiseq 2500, by Illumina (1 mentions) Truseq rna sample preparation v2 low sample protocol guide, by Illumina (1 mentions) Tapestation 2200, by Agilent Technologies (1 mentions) Genome analyzer gaii, by Illumina (1 mentions) Qubit rna assay kit, by Thermo Fisher Scientific (1 mentions) Truseq rna sample preparation kit, by Illumina (1 mentions) Invitrap spin cell rna mini kit, by Stratec (1 mentions)

7 protocols using truseq rna sample prep v2 ls protocol

Comparative Transcriptome Profiling Using Microarray and RNA-Seq

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RNA was harvested from each of the four samples (row 2 of Figure 1). The four RNA samples were then processed twice on different days using the Agilent Quick Amp Labeling kit (Part no. 5190-0424) to produce eight cRNA libraries, each of which was then hybridized on two separate chips (Yeast Expression 8 × 15K arrays) on different days according to the factorial design. After the arrays were washed and scanned, features were extracted using the Agilent Feature Extraction software to determine red and green intensities.
The same four RNA samples were also processed using the Illumina TruSeq RNA Sample Prep v2 LS protocol. Each sample was prepared twice up to the 3′ end adenylation step, then each of the eight preparations was split into two aliquots, after which each was indexed and amplified to complete the RNA-Seq library preparation (row 3 of Figure 1). The two groups of eight samples that were indexed together were each mixed at equimolar concentrations and sequenced on separate lanes on the same Illumina HiSeq 2500 flowcell, to produce 141 bp reads. In total, we produced profiles from 16 RNA-Seq samples and 16 microarray samples with identically nested experimental designs (row 4 of Figure 1).

Robinson D.G., Wang J.Y, & Storey J.D. (2015). A nested parallel experiment demonstrates differences in intensity-dependence between RNA-seq and microarrays. Nucleic Acids Research, 43(20), e131.

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mRNA Library Preparation for Illumina Sequencing

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The mRNA libraries were prepared from 16 samples of total RNA in 2 biological replicates for most of the tissues according to the TruSeq RNA sample Prep v2 LS protocol (Illumina, San Diego, CA, U.S.A.). Briefly, mRNA was purified from the total RNA samples using poly-T oligo-attached magnetic beads followed by mRNA fragmentation, first- and second-strand cDNA synthesis. Later, the overhangs resulting from the fragmentation of double-stranded (ds) cDNA were repaired to form blunt ends. A single “A” nucleotide was added to the 3′ ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. Multiple indexing adapters were ligated to the ends of ds cDNA to prepare them for hybridization onto a flow cell followed by a PCR amplification step. The libraries were quantified using the qPCR technique and analyzed on a Bioanalyzer 2100 (Agilent Technologies) using a DNA specific chip. Subsequently, the libraries were normalized and pooled together followed by flow-cell cluster generation using a cBot fully automated clonal cluster generation system for Illumina sequencing. Single-end multiplexed sequencing was done using the Illumina GAIIx platform with the total of 100 cycles.

Bilichak A., Ilnytskyy Y., Wóycicki R., Kepeshchuk N., Fogen D, & Kovalchuk I. (2015). The elucidation of stress memory inheritance in Brassica rapa plants. Frontiers in Plant Science, 6, 5.

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Transcriptomic Profiling of Pistil Development

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Based on microscopy inferences, 48 HAP pistils of SP_S, SP_T and CP (SxT) in ten biological replicates were collected and snap-frozen to liquid nitrogen for total RNA extraction using IRIS method [51 (link)]. The RNA was quantified on NanoDrop 2000 (Thermo Scientific, Waltham, MA, USA), and quality was assessed on 1% formaldehyde agarose gel (MOPS) and Agilent Bioanalyzer with RNA 7500 series II Chip (Agilent Technologies, CA, USA). The RNA samples with RIN (RNA Integrity Number) value greater than 8 and the final concentration of 4.0 µg were used for cDNA library preparation.
Eight cDNA libraries in biological replicates SP_T (3), CP (3) and SP_S (2) were constructed using the illumina Truseq RNA Sample prep v2 LS Protocol (Illumina Inc., CA, USA). The libraries were quantified on Qubit 2.0 fluorometer (Invitrogen, USA), while quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). The paired-end (PE) (2 × 72 bp) sequencing was performed using Illumina GAIIx.

Seth R., Bhandawat A., Parmar R., Singh P., Kumar S, & Sharma R.K. (2019). Global Transcriptional Insights of Pollen-Pistil Interactions Commencing Self-Incompatibility and Fertilization in Tea [Camellia sinensis (L.) O. Kuntze]. International Journal of Molecular Sciences, 20(3), 539.

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Measuring Global mRNA Half-Lives in Cell Lines

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For global mRNA half-life measurements, MCF7 and HEK293 cells were labeled with 700 μM 4SU for 60 minutes. Total RNA was extracted using the miRNeasy kit (QIAGEN). 4SU residues were biotinylated using EZ-Link biotin-HPDP (Thermo Fisher Scientific (Waltham, MA, USA)). Biotinylated 4SU-labled RNA was separated from non-labeled RNA using μMACS Streptavidin MicroBeads (Miltenyi (Bergisch Gladbach, Germany)) and 4SU-labeled RNA was eluted from μColumns by addition of 100 mM DTT. RNA was recovered from the flow-though and 4SU-labeled fractions using MinElute Spin columns (QIAGEN). Input (total), flow-though (non-labeled RNA) and eluted (4SU-labled RNA) samples were used for poly(A) + mRNA library preparation following the TruSeq RNA sample Prep v2 LS protocol (Illumina). The libraries were sequenced on an Illumina Hiseq 2500 for 100 cycles (multiplexed 1 × 101 + 7 index). mRNA half-lives were computed from gene-wise FPKM (fragments per kilobase of exonic sequence per million fragments mapped) as previously described [51 (link)]. To access changes in mRNA half-life, we computed the log2 fold change of all measured genes on quantile normalized data.
MCF7 and HEK293 half-life measurement sequencing data have been deposited under GEO accession number GSE49831.

Schueler M., Munschauer M., Gregersen L.H., Finzel A., Loewer A., Chen W., Landthaler M, & Dieterich C. (2014). Differential protein occupancy profiling of the mRNA transcriptome. Genome Biology, 15(1), R15.

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Illumina TruSeq RNA Sequencing Protocol

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For each genotype, 1 μg of RNA from each of the four biological replicates was pooled (4 μg) into a single sample and used for cDNA library preparation. The cDNA libraries were prepared following the TruSeq RNA Sample Preparation v2 low sample (LS) protocol guide (Illumina Inc.). Poly(A)-containing mRNA was purified twice using poly(T) oligonucleotide-attached magnetic beads. In the second elution, the Poly(A) RNA was fragmented and primed for cDNA synthesis at 94°C in an attempt to obtain a median insert size of 180 bp fragment. The fragmented RNA templates were primed with random hexamers, and the first strand was synthesized by four cycles of 25°C for 10 minutes, 42°C for 50 minutes, and 70°C for 15 minutes. Following second strand synthesis (16°C for one hour), end repair was performed to generate blunt ends followed by adenylating of the 3′ blunt-ended double-stranded cDNAs to allow for subsequent ligation of multiple indexing adaptors. cDNA fragments were amplified and enriched using 15 cycles of PCR according to Illumina TruSeq RNA Sample Prep v2 LS protocol. The libraries were quantified using a Qubit^® 2.0 Fluorometer (Life Technologies), and the quality was analyzed with the TapeStation 2200 (Agilent) using the D1K tape for validating the purity and estimating the insert size.

Kottapalli P., Ulloa M., Kottapalli K.R., Payton P, & Burke J. (2016). SNP Marker Discovery in Pima Cotton (Gossypium barbadense L.) Leaf Transcriptomes. Genomics Insights, 9, 51-60.

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MCF7 and HEK293 mRNA-seq protocol

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MCF7 cells were maintained at 37°C in RPMI supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin. For RNA isolation, 5 × 105 cells were grown in triplicates under normal conditions, and harvested two days later. Cells were lysed and RNA was isolated using the InviTrap Spin Cell RNA Mini Kit (Stratec Molecular GmbH (Berlin, Germany)). RNA quality was analyzed with the Agilent RNA 6000 Nano Kit, and the concentration was measured with the Qubit RNA Assay Kit (Invitrogen). Library preparation was carried out with the TruSeq™ RNA Sample Preparation Kit (Illumina (San Diego, CA, USA)) using barcoded primers. Libraries were sequenced on Illumina HiSeq using a paired-end protocol (2 × 100 nucleotides).
MCF7 mRNA-seq sequencing data have been deposited under GEO accession number GSE49831.
HEK293 total RNA was extracted using the miRNeasy kit (Qiagen (Hilden, Germany)) following the instructions of the manufacturer. RNA (4 μg) was used for poly(A) + mRNA library preparation following the TruSeq RNA sample Prep v2 LS protocol (Illumina). The libraries were sequenced on an Illumina Genome Analyzer GAII or Illumina HiSeq for 100 cycles (multiplexed 1 × 101 + 7 index).
HEK293 mRNA-seq sequencing data have been deposited under GEO accession number GSE49831.

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Preparing RNA-seq Libraries for MiSeq

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A total of six samples, three replicates for each strain, were used to generate cDNA libraries using 500 ng of total RNA and following the TruSeq RNA Sample Prep v2 LS Protocol (Illumina, CA, USA). Pooled libraries were normalized to 11 pM, and 1% v/v of 11 pM PhiX sequencing control was added. A paired-end sequencing run was performed (2x81 bp) using the MiSeq Reagent Kit v3 in the MiSeq Sequencer (Illumina, USA).

Mora-Godínez S., Senés-Guerrero C, & Pacheco A. (2024). De novo transcriptome and lipidome analysis of Desmodesmus abundans under model flue gas reveals adaptive changes after ten years of acclimation to high CO2. PLOS ONE, 19(5), e0299780.

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