K562 cells were grown as described previously. At approximately 50% confluence, the media was supplemented with s4U (100 μM). The cells were incubated at 37°C for 4h, at which point total RNA was isolated using the RNeasy mini kit with the following modifications: buffers RLT and RPE were supplemented with 1% final 2-mercaptoethanol (BME); an additional 80% EtOH wash was performed after the RPE step; and the column was spun at maximum speed for 5 min to dry prior to elution with water. The isolated RNA was then chemically treated and purified as described previously. For each sample, 10 ng of total RNA was used to construct a sequencing library using the Clontech SMARTer Stranded Total RNA-Seq kit (Pico Input) with ribosomal cDNA depletion. Paired-end 150bp sequencing was performed on an Illumina HiSeq 4000 instrument. TimeLapse-seq was performed in duplicate using biologically distinct samples for experimental samples. Raw and processed sequencing data have been submitted to the GEO database.
Smarter stranded total rna seq kit
Manufactured by Takara Bio
Sourced in United States, Japan
The SMARTer Stranded Total RNA-Seq Kit is a lab equipment product designed for total RNA-Seq library preparation. It enables efficient capture and sequencing of full-length, stranded RNA transcripts from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (6 mentions)
Nextseq 500, by Illumina (5 mentions)
Novaseq 6000, by Illumina (5 mentions)
Rnaeasy plus micro kit, by Qiagen (4 mentions)
Pico input mammalian, by Takara Bio (4 mentions)
D1000 screentape, by Agilent Technologies (4 mentions)
Qubit rna assay kit, by Thermo Fisher Scientific (4 mentions)
Hiseq 4000, by Illumina (3 mentions)
Hiseq platform, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Rneasy micro kit, by Qiagen (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Rneasy plus kit, by Qiagen (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nextseq 500 desktop next generation sequencing system, by Illumina (2 mentions)
Profiler pcr array, by Qiagen (2 mentions)
Nextseq 500 platform, by Illumina (2 mentions)
Truseq pe cluster kit v3 cbot hs, by Illumina (2 mentions)
Epimark n, by New England Biolabs (2 mentions)
36 protocols using smarter stranded total rna seq kit
1
s4U Labeling and RNA-seq in K562 Cells
2
RNA-Seq Analysis of Sorted Cell Populations
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Total RNA was extracted from flow cytometry–sorted cells using QIAshredders and an RNeasy Plus Micro kit (QIAGEN) (25 (link), 45 (link)). RNA quality and quantity were analyzed using a NanoDrop and Bioanalyzer. RNA-Seq library preparation and sequencing were conducted at the Genomics and Microarray Core at the University of Colorado Anschutz Medical Campus (46 (link)). Libraries were constructed using a SMARTer Stranded Total RNA-Seq kit (Clontech) customized with mouse-specific oligonucleotides for rRNA removal. Directional mRNA-Seq of sorted cell populations was conducted using the Illumina HiSeq 2500 or 4000 system.
3
Transcriptomic Analysis of Plaque and pWAT
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pWAT FBC, FB RNA was extracted with RNeasy micro kit (Qiagen), plaque and pWAT T cells RNA was extracted with PicoPure RNA isolation kit (Thermo Fisher scientific), and quality and quantity were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA seq libraries were prepared using the Clontech SMARTer Stranded Total RNA Seq Kit - Pico Input Mammalian following the manufacturer’s protocol. Libraries were purified using AMPure beads, pooled equimolarly, and run on a HiSeq 2500 (plaque CD68+ samples) or HiSeq 4000 (Plaque T cells and pWAT FBC, FB and T cells samples), paired end reads. FASTQ files were obtained, and low-quality bases as well as adapter sequences were trimmed using Cutadapt 1.18. Reads were subsequently mapped to the Mus musculus GRCm38 transcriptome using Kallisto 0.46.2. Raw counts per transcript were summed up at the gene level, and differential expression analysis was performed using edgeR 3.28.0. Genes with a P value < 0.05 and logFC > 0.6, or FDR < 0.05 and logFC > 1 between conditions were determined to be differentially expressed. Heatmaps were generated using heatmap 1.0.12. Pathways analysis was performed using Ingenuity Pathway Analysis (IPA, Qiagen).
4
s4U Labeling and RNA-seq in K562 Cells
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K562 cells were grown as described previously. At approximately 50% confluence, the media was supplemented with s4U (100 μM). The cells were incubated at 37°C for 4h, at which point total RNA was isolated using the RNeasy mini kit with the following modifications: buffers RLT and RPE were supplemented with 1% final 2-mercaptoethanol (BME); an additional 80% EtOH wash was performed after the RPE step; and the column was spun at maximum speed for 5 min to dry prior to elution with water. The isolated RNA was then chemically treated and purified as described previously. For each sample, 10 ng of total RNA was used to construct a sequencing library using the Clontech SMARTer Stranded Total RNA-Seq kit (Pico Input) with ribosomal cDNA depletion. Paired-end 150bp sequencing was performed on an Illumina HiSeq 4000 instrument. TimeLapse-seq was performed in duplicate using biologically distinct samples for experimental samples. Raw and processed sequencing data have been submitted to the GEO database.
5
Diverse RNA Library Preparations for Viral Sequencing
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Two different RNA library-priming approaches (random-hexamer priming and template-switching reverse transcription) were used. Two 150 bp paired-end libraries (cDNA from total RNA) were generated using random-hexamer priming with the TruSeq RNA Library Prep Kit (Illumina) for the virus RNA extracts with or without DNase I digestion. Two single-end libraries were generated for the DNase I treated viral RNA extract using template-switching reverse transcription with the SMARTER stranded total RNA-seq kit (Clontech): one without fragmentation, and one with 4 min fragmentation at 94 °C, according to manufacturer’s instructions. The TruSeq Nano DNA HT Library Prep Kit (Illumina) was used to generate a 150-bp paired-end DNA library from the virus DNA extracts (Extended Data Fig. 8 ). High-throughput sequencing was performed on the Illumina MiSeq platform with v3 chemistry, and subsequently on the Illumina HiSeq 2500 platform. Both the library preparation and high-throughput sequencing were performed by Biozeron (Shanghai). Sequencing parameters are shown in Extended Data Fig. 9 .
6
Mammalian Total RNA-seq with ZAPr
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RNA-seq libraries were prepared at the MIT BioMicroCenter using the following kit: Clontech SMARTer Stranded Total RNA-Seq Kit—Pico Input Mammalian with ZAPr ribosomal depletion. Paired-end sequencing was performed using the 150nt Nextseq kit.
7
Comprehensive RNA-Seq Analysis Pipeline
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Total RNA was extracted from cell lines using RNeasy Mini Kits (Qiagen). The isolated RNA was processed and prepared using the SMARTer® Stranded Total RNA-Seq Kit (Clontech) according to the manufacturer’s protocol. Libraries were sequenced on Illumina NextSeq using paired-end 150-bp reads. In order to remove low-quality bases, we first optimized the trimming length using mapping rate and depth of coverage (data not shown). Then, the original 2×150 paired-end reads were trimmed to 2×100 reads. We then used STAR 2.3 to align the reads to the human genome transcriptome65 . We used RSEM 1.2.29 package to calculate the gene-level FPKM values66 (link). Differentially expressed genes were found by DESeq2 R package67 (link). Next, in order to analyze pathway enrichment, we utilized the GSEA suit (in “weighted” Enrichment statistic mode) with the inferred coverage-based ranked set68 (link) (ranked by fold change).
8
Extracellular Vesicle RNA Profiling Protocol
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Extracellular vesicle‐derived long RNAs isolated from plasma were treated with DNase I (NEB) to remove DNA. Strand‐specific RNA‐seq libraries were then prepared using the SMARTer Stranded Total RNA‐seq kit (Clontech). Library quality was analyzed using a Qubit Fluorometer (Thermo Fisher Scientific) and Qsep100 (BiOptic); 2 × 150 bp paired‐end sequencing was performed on an Illumina sequencing platform. The raw sequencing reads were filtered by FastQC (version 0.11.8) and aligned to the Gencode human genome (GRCh38) using the read aligner STAR (version 2.7.1a).28 Gene expression levels were then quantified by featureCounts (version 1.6.3)29 and transformed into transcripts per kilobase million (TPM). Annotation information of mRNA, lncRNA, and pseudogene genes were retrieved from the GENCODE database (Human, version 29). The circRNAs were discovered and quantified by the Assembling Splice Junctions Analysis software.30
9
RNA-sequencing of Pyramidal and PV Neurons
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Generation of microarray data from pyramidal and PV neurons was previously described [25 (link),26 (link)]. For RNAseq, RNA was isolated using RNeasy micro kits (Qiagen), and cDNA libraries were prepared using the pico input SMARTer stranded total RNA-seq kit (Clontech). Library size was measured by high sensitivity DNA kit (Agilent), and concentration was quantified by Qubit (Life Technologies). Libraries were sequenced on a NextSeq500 (Illumina), and reads were aligned to the human genome using SALMON [27 (link)], and differential expression of genes between cell types was quantified using DESEQ2. On average, 5 million reads mapped to annotated gene regions for each sample. The number of genes with TPM (transcripts per million) >1 was 12,000–13,000 for the isolated cell types, and over 16,000 for slide controls. All data have been deposited to GEO (GSE149154).
10
RNA-seq Analysis of Transcriptome Profiling
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RNA-seq analysis was performed as previously described (31 (link)). Total RNAs were purified using an RNeasy Plus Kit (Qiagen). RNA-seq libraries were prepared using a SMARTer Stranded Total RNA-seq Kit (Clontech) and sequenced as 101 bp paired-end reads on a HiSeq 2500 or NovaSeq 6000 (Illumina). RNA-seq reads were processed using Trimmomatic and aligned to the human reference genome (hg19). Gene expression levels were quantified as fragments per kilobase of transcript per million mapped reads (FPKM). Differentially expressed genes were determined with the cut-off of FDR < 0.05 and fold change > 1.5. Volcano, scatter and violin plot analyses were performed using R software package.
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