Given the minimal amount of RNA obtained from IVLE, the Smart-Seq2 protocol was adapted for low-input RNA library preparation (25 (link)). Two different amounts of input RNA, 12 ng and 3 ng, for each sample and its replicates were prepared. Polyadenylated mRNA was enriched using 5 mg/ml Dynabead Oligo(dT)20 in 1X PBS (pH 7.4) from mRNA DIRECT kit (Thermo Fisher). Beads were washed with 10mM Tris-HCl pH 7.5, 150 mM LiCl, 1mM EDTA pH 8.0, 0.1% w/v LiDS in nuclease free water and RNA eluted using 10 mM Tris-HCl pH 8.0 at 75°C for 2 minutes. The poly A-enriched RNA was reverse transcribed by Smart-Seq2 as described (25 (link)) with 10 reverse transcriptase cycles, and cDNA further amplified using IsoSeq PCR (ISPCR) primers with 10 or 11 PCR cycles. Dual indexed sequencing libraries were made out of 5 ng cDNA from the above preparations using Illumina Nextera library preparation kit according to manufacturer’s instructions (Illumina). Quality checked and equimolar pooled libraries were sequenced in a HiSeq 4000 Illumina system and 75bp paired- non-stranded- reads generated. Sequence data were deposited in the European Nucleotide Archive (ENA) with the study number ERP128933; NCBI BioProject ID PRJEB44842 (accession numbers for each sample are shown in Supplementary Table S1 ).
Mrna direct kit
Manufactured by Thermo Fisher Scientific
The MRNA Direct kit is a laboratory equipment product designed for the extraction and purification of mRNA from biological samples. The kit provides a streamlined and efficient method for isolating mRNA, which can be used for various downstream applications, such as gene expression analysis and mRNA-based research.
Automatically generated - may contain errors
Lab products found in correlation
Superscript 3 rt enzyme, by Thermo Fisher Scientific (3 mentions)
Hiseq 4000 system, by Illumina (2 mentions)
Dynabead oligo dt 20, by Thermo Fisher Scientific (2 mentions)
Nextera library preparation kit, by Illumina (2 mentions)
Tapestation 4200, by Agilent Technologies (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Actinomycin d, by Thermo Fisher Scientific (1 mentions)
Spin x, by Corning (1 mentions)
Nuclease p1, by Merck Group (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Dragen bio it, by Illumina (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Hiseq 3000, by Illumina (1 mentions)
Sequencing adapter, by Illumina (1 mentions)
Oligo dt beads, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
9 protocols using mrna direct kit
1
Smart-Seq2 Low-Input RNA Library Prep
2
Isolation of EBOV-Infected Cell RNA
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NegCtrl and METTL3 KO cells were seeded in 6-well plates and infected one day later with rgEBOV (MOI = 1). 24 hpi, medium was exchanged against DMEM with 5% FCS containing 0.02 mg/ml Actinomycin D (Gibco), and 48 hpi supernatant was removed and cells were washed in 1 ml PBS per well. After centrifugation for 5 min at 1000 x g cell pellets were resuspended in 250 µl PBS and transferred to tubes containing 750 µl TRIzol (Thermo Fisher Scientific) and removed from the BSL4 laboratory. RNA was isolated according to the manufacturer’s instructions. mRNA was further isolated using the mRNA DIRECT Kit (Thermo Fisher Scientific) following the manufacturer’s instructions, and subsequently used for miCLIP analysis without fragmentation as described previously [26 (link)]. For further details see Supplemental methods.
3
Smart-Seq2 Low-Input RNA Library Prep
4
Large-scale mRNA Purification and Analysis
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For large-scale mRNA extractions, polyadenylated mRNA was enriched using 100 µl oligo dT agarose beads (NEB) per mg RNA. RNA was bound to the beads in binding buffer (1 M NH4OAc, 2 mM EDTA) for 5 min rotating at room temperature and washed once. RNA was eluted in room temperature water, filtered in SpinX (Corning) tubes to remove any beads, then precipitated in 2.5 M NH4OAc and isopropanol, centrifuged at 14 000 r.p.m. × 30 min and pellets washed in 75% ethanol.
To extract mRNA from HeLa cell samples an mRNA Direct kit (ThermoFisher) was used. Magnetic oligo dT beads (40 µl) were used per sample, but with the binding buffer and incubation times indicated above. mRNA was digested with 9.5 units of Nuclease P1 (Sigma) in 20 mM NH4OAC pH 5.3 for 3 h at 37°C. Then, 250 fmol of ARCA was added, and LC–MS was carried out as described above.
To extract mRNA from HeLa cell samples an mRNA Direct kit (ThermoFisher) was used. Magnetic oligo dT beads (40 µl) were used per sample, but with the binding buffer and incubation times indicated above. mRNA was digested with 9.5 units of Nuclease P1 (Sigma) in 20 mM NH4OAC pH 5.3 for 3 h at 37°C. Then, 250 fmol of ARCA was added, and LC–MS was carried out as described above.
5
Oleic Acid Exposure Regulates PLIN2 in RPTECs
6
RNA-Seq Analysis of JW23.3 Cell Responses
7
Yeast Transcriptome Analysis by RNA-seq
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Yeast cultures were grown to OD600 0.5 in 50 ml of a medium containing 1.0% yeast extract, 2.0% peptone, 2.0% raffinose, and 0.1% glucose. Expression of proteins was induced by adding galactose to 2% into the cultures and incubating the cultures at 30° for an additional 60 min. Cells were collected by centrifugation, and the pellets were scraped with spatula, flash-frozen in liquid nitrogen, and stored at −80°. RNA was isolated using mRNA DIRECT kit (Life Technologies) according to manufacturer’s instructions. Construction of RNA-seq libraries was performed as previously described (Labunskyy et al. 2014 (link)), and the libraries were sequenced on the Illumina HiSeq2000 platform. RNA-seq reads were aligned to the S. cerevisiae genome from the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org/ , release number R64-1-1) and sequences of the introduced exogenous genes. Sequence alignment was performed using Bowtie software v.0.12.7 (Langmead et al. 2009 (link)) allowing two mismatches per read. To analyze expression of exogenous genes, rpkm (reads per kilobase per million mapped reads) values, which represent the number of reads normalized to gene length and total number of mapped reads, were calculated for each gene. An average rpkm value for two biological replicates is shown.
8
Illumina-Based RNA Sequencing Protocol
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RNA sequencing was performed by the Washington University Genome Technology Access Center) as we reported51 (link). Library preparation was performed with 10 μG of total RNA with a Bioanalyzer RIN score >8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct kit, Life Technologies). mRNA was fragmented and reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies) and random hexamers. A second strand reaction was performed to yield ds-cDNA, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were amplified for 12 cycles and sequenced on an Illumina HiSeq-3000 using single reads extending 50 bases. RNA-seq reads were aligned to the Ensembl release 76 top-level assembly with STAR version 2.0.4b. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 1.4.5. Transcript counts were produced by Sailfish version 0.6.3.
9
Transcriptional Profiling of JW23.3 Cells
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JW23.3 cells were treated with 40 μmol/L WU-12a, 40 μmol/L WU-76, or vehicle control (DMSO) for 48 hours. RNA was isolated as described above, and samples contained at least 5 μg of purified total RNA with RIN >9.0. At least three biological replicates were performed. Samples were aligned against Mouse Ensembl GRCm38.76. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 5 to 10 μg of total RNA with a Bioanalyzer RIN score greater than 8.0. Ribosomal RNA was removed by poly-A selection using Oligo-dT beads (mRNA Direct Kit, Life Technologies). mRNA was then fragmented in reverse transcriptase buffer and heating to 94° for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3′ ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12 to 15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases. Differential expression analysis was performed with the DESeq2 package (R Bioconductor software; RRID:SCR_015687).
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