NRCMs were harvested, after cultivation in DMEM-F12 containing 10% FCS for 24 h and in DMEM-F12 in 0.5% FCS for 47 h, in ice cold mammalian polysome buffer (10 mM MgCl, 20 mM Tris pH 7.4, 2 mM DTT, 200 mM KCl, 1% Triton X-100,) containing 40 U/µl Murine RNAse inhibitor, and1 x protease inhibitor cocktail and sonicated for complete lysis. Lysate was incubated with Ybx1 antibody (D2A11) overnight at 4 °C, then for 1 h at RT with prewashed sheep anti rabbit IgG Dynabeads (ThermoFisher). Coprecipitate was washed once with ice cold wash buffer (polysomal buffer with 10% Triton, DNase I), three times with ice cold high salt buffer (polysomal buffer with 1 M KCl, 10% Triton, DNase I) and then once finally with wash buffer. Samples were divided for use for protein or RNA. Protein elution was performed by denaturing the samples with laemmli buffer at 95 °C for 5 min.RNA was eluted using Trizol and following extraction using chloroform. Library generation was done using the Lexogen Quant-Seq kit according to the manufacture’s instruction. Libraries were then multiplexed and sequenced on a NextSeq550.
Quantseq kit
Manufactured by Lexogen
Sourced in Austria
The QuantSeq kit is a library preparation kit designed for generating sequencing-ready libraries from total RNA samples. It enables the efficient and accurate quantification of gene expression levels through 3' mRNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 4000, by Illumina (4 mentions)
Hiseq 4000 sequencer, by Illumina (2 mentions)
Novaseq sequencer, by Illumina (2 mentions)
Novaseq 6000, by Lexogen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nextseq 500, by Illumina (1 mentions)
Ercc rna spike in mix 1, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Minelute column, by Qiagen (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
Iodoacetamide, by Thermo Fisher Scientific (1 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Tapestation 2200, by Agilent Technologies (1 mentions)
Qubit system, by Thermo Fisher Scientific (1 mentions)
7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Kapa library quantification kit, by Avantor (1 mentions)
Nextseq sequencer, by Illumina (1 mentions)
Nextera flex kit, by Illumina (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Nucleospin rna kit, by Macherey-Nagel (1 mentions)
13 protocols using quantseq kit
1
Transcriptome Analysis of Cardiomyocyte RNA-Binding
2
Auxin-Induced Transcriptional Dynamics
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Cell were treated for 6 h with auxin and then with 800 μM 4sU (Sigma-Aldrich) for 0, 1, 2 or 4 h. Then, cells were harvested in RLT buffer and RNA was extracted using RNEasy kit (QIAGEN) with on-column DNAase digestion (QIAGEN). ERCC RNA Spike-in Mix 1 (Thermo Fisher Scientific) was added to each RNA sample in equal amounts, alkylation was carried out using 10 mM iodoacetamide (Thermo Fisher Scientific), and the reaction was quenched with 0.1 M DTT (Thermo Fisher Scientific). Alkylated RNA was purified on MinElute columns (QIAGEN). RNA integrity was verified using standard RNA kit on Fragment Analyzer (Agilent Technologies). For samples passing quality checks, the RNA was subjected to library preparation using QuantSeq kit (Lexogen) for 13 cycles and sequenced for 75 cycles on Illumina NextSeq500.
3
RNA-Seq Analysis of Murine Tumor Tissue
4
Transcriptional Analysis of Spinal Cord Development
5
Differential Gene Expression Analysis of Dinaciclib-Treated HD-MB03 Cells
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HD-MB03 cells were treated with DMSO or 1 nM dinaciclib for 24 h. All treatment conditions were submitted and processed in triplicates at the Sequencing facility of the Cancer Research, UK. Sequencing libraries were prepared by using the QuantSeq kit (Lexogen) and run on an Illumina NextSeq sequencer.
A sequential alignment procedure was performed to map raw reads against the GRCh38 Human Genome reference (release 83). First, all reads were aligned by ‘STAR’54 (link); then, using ‘Bowtie2’55 (link), we locally mapped those reads discarded by STAR. Gene expression quantification was computed by ‘featureCounts’56 (link). The ‘DaMiRseq’ Bioconductor/R package was used to filter out genes (less than 10 counts in more than 50% of samples), perform the normalization (variance stabilizing transformation), and search for confounding factors57 (link). Differential analysis was performed by the ‘limma’ R/Bioconductor package58 (link). A gene was deemed significant whether the P value was < 0.05 and logFC threshold of 0.5. A Benjamini–Hochberg procedure was performed to control the false discovery rate (FDR).
A sequential alignment procedure was performed to map raw reads against the GRCh38 Human Genome reference (release 83). First, all reads were aligned by ‘STAR’54 (link); then, using ‘Bowtie2’55 (link), we locally mapped those reads discarded by STAR. Gene expression quantification was computed by ‘featureCounts’56 (link). The ‘DaMiRseq’ Bioconductor/R package was used to filter out genes (less than 10 counts in more than 50% of samples), perform the normalization (variance stabilizing transformation), and search for confounding factors57 (link). Differential analysis was performed by the ‘limma’ R/Bioconductor package58 (link). A gene was deemed significant whether the P value was < 0.05 and logFC threshold of 0.5. A Benjamini–Hochberg procedure was performed to control the false discovery rate (FDR).
6
HepG2 Transcriptome Profiling by RNA-seq
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Total RNA was extracted from HepG2 cells using trizol (ThermoFisher) followed by RNA column purification (Purelink Invitrogen) and on-column DNA digestion (Purelink, Invitrogen). RNA was used to create 3′ end specific libraries with the Quant Seq kit (Lexogen). The same sample of HepG2 RNA was depleted of ribosomal RNA (Ribominus Eukaryotic v2, Fisher) and used to generate standard Illumina short-read libraries using the Nextera Flex kit (Illumina). Both libraries were sequenced (Illumina MiSeq, 300 kit) and the fastq sequence files used in downstream analysis. The standardized Bluebee Lexogen Quant Seq FWD protocol was used for trimming, read alignment, and quality control steps of the Quant Seq library. The standard HepG2 transcriptome sequencing was analyzed in the same manner as other fastq datasets (see—Calculation of SERPINA1 distal ratio from sequencing).
7
KRAS Variant RNA-seq Profiling
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For RNA sequencing, 70,000 cells per well (6-well plate) of either KRASWT or KRASG12D were seeded 24 h in advance. The next day, cells were washed with PBS and RNA was isolated using the NucleoSpin RNA kit (740955.5, Macherey-Nagel) according to the manufacturer’s instructions. cDNA libraries amplified from the 3′ UTR were generated from total RNA using the Lexogen QuantSeq kit (Lexogen, Austria) according to the standard protocol and sequenced with a 50-bp single-end protocol on Illumina HiSeq4000 sequencer (Illumina, USA). The raw-sequencing data was aligned to the respective mouse reference genomes and quantified prior to differential expression analyses. Raw FPKM values of each transcript were transformed by log2 (FPKM + 0.01). Data processing and statistical analyses were performed using Microsoft Excel (Microsoft, USA) and Instant Clue software [17 (link)] which performed a hierarchical clustering to classify the experiments and generate a heatmap for the visualization of different RNA expression.
8
Transcriptome sequencing of PDAC models
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Transcriptome sequencing was generated during this study for patient-derived cell lines and samples and the lung adenocarcinoma mouse models were performed using total extracted RNA. 3′ UTR mRNA libraries were generated from total RNA using the Lexogen QuantSeq kit (Lexogen, Austria) according to the standard protocol and sequenced on Illumina HiSeq4000 or NovaSeq sequencer (Illumina, USA). RNA extracted from primary murine PDAC cell lines was processed using TruSeq Stranded mRNA Kit (Illumina, USA) and sequenced as 2 × 100 bp on a HiSeq 4000 (Illumina, USA). Raw-sequencing data were aligned to the respective reference genomes and quantified prior to differential expression analyses. For details see Supplementary Materials and Methods.
9
IL-33 Isoform Effects on HRMVECs
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Quiescent HRMVECs were treated with IL-33 95-270 , IL-33 99-270 , IL-33 109-270 and IL-33 112-270 for 24 h. TRIzol reagent was used to extract total cellular RNA, which was then sent for mRNA sequencing. Gene expression was determined by creating 3′ mRNA-seq libraries with Lexogen's Quant-seq Kit and sequencing (1 × 75 bp) on a NovaSeq 6000. STAR was used to align sequencing data to the mouse genome (mm10) before tabulating counts across genes (htseq-count). EdgeR was used to identify differentially expressed genes, and heatmaps were created using the R program's WGCNA package 29 (link) and modifying the R-codes according to a method outlined by Johnstone 30 . The mRNA sequencing data were uploaded to the Gene Expression Omnibus (code GSE245784).
10
Retina RNA-seq of OIR mice
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At P15, the normoxic and OIR-exposed C57BL/6 mice pups were euthanized, retinas isolated, total cellular RNA was extracted by TRIzol reagent, and sent for mRNA sequencing. Gene expression was identified using 3’mRNA-seq libraries generated from Lexogen’s Quant-seq kit before sequencing (1 × 75 bp) on a NovaSeq 6000. Sequencing data was aligning to the mouse genome (mm10) using STAR before tabulating counts across genes (htseq-count). Differentially expressed genes were determined in R using edgeR with heatmaps generated using the pheatmap package. Significantly altered genes (|log fold change| = 2; p-value = 0.05) were selected for Gene Ontology analysis (RDAVIDWebService). The mRNA sequencing data has been submitted to the Gene Expression Omnibus (code GSE209654) and is now available to the public.
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