Frozen muscle biopsies were homogenized in 20 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT and 1% Triton X-100, and total RNA was extracted from using TRIzol® LS reagent (Life Technologies) followed by isopropanol precipitation according to the manufacturer’s instructions. Cytoplasmic and mitochondrial rRNA was depleted from ~ 1 μg of total RNA using tiling oligodeoxynucleotides and the RNase H digestion method [56 (link)]. Illumina TruSeq Stranded Total RNA library kits were used to prepare random-primed, indexed RNA-seq libraries from the rRNA-depleted total RNA preparations and these libraries were sequenced on an Illumina HiSeq 2500 instrument using single-end 50 bp v4 read chemistry. Quality metrics for FASTQ reads were evaluated with FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc ) and reads were quality trimmed and adapter clipped using the Trimmomatic tool [57 (link)] with standard parameters. FASTQ sequence reads were mapped to the Broad CanFam3.1/canFam3 (Sep. 2011) reference genome using the STAR v2.7 RNA-seq aligner [58 (link)]. Strand-specific coverage plots were generated from the alignment files using the BEDTools genomecov function and displayed in the UCSC genome browser after conversion with the UCSC bedGraphToBigWig utility.
Truseq stranded total rna library kit
Manufactured by Illumina
Sourced in United States
The TruSeq Stranded Total RNA library kit is a laboratory equipment product designed for preparing total RNA samples for next-generation sequencing. The kit enables the conversion of total RNA into a library of cDNA fragments with adapter sequences attached to both ends, allowing for sequencing on Illumina platforms.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (4 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (2 mentions)
Quick rna microprep kit, by Zymo Research (2 mentions)
Novaseq sp reagent kit, by Illumina (2 mentions)
Qiaamp viral rna mini, by Qiagen (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Hiseq 2500 instrument, by Illumina (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Omni tissue homogenizer, by Omni International (1 mentions)
Sureselect xt library preparation kit, by Agilent Technologies (1 mentions)
Ez1 dsp virus kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Truseq small rna sample prep kit, by Illumina (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Mirvana isolation kit, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
19 protocols using truseq stranded total rna library kit
1
Transcriptome Profiling of Frozen Muscle Biopsies
2
RNA-Seq Analysis of Muscle Biopsies
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Frozen muscle biopsies were homogenized in 20 mM Tris HCl pH 7.4, 150 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, and 1% Triton X-100, and total RNA was extracted from using TRIzol® LS reagent (Life Technologies) followed by isopropanol precipitation according to the manufacturer's instructions. Cytoplasmic and mitochondrial rRNA was depleted from ~1 microgram of total RNA using tiling oligodeoxynucleotides and the RNase H digestion method [55] . Illumina TruSeq Stranded Total RNA library kits were used to prepare random-primed, indexed RNA-seq libraries from the rRNA-depleted total RNA preparations and these libraries were sequenced on an Illumina HiSeq 2500 instrument using singleend 50 bp v4 read chemistry. Quality metrics for FASTQ reads were evaluated with FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc) and reads were quality trimmed and adapter clipped using the Trimmomatic tool [56] with standard parameters. FASTQ sequence reads were mapped to the Broad CanFam3.1/canFam3 (Sep. 2011) reference genome using the STAR v2.7 RNA-seq aligner [57] . Strand-speci c coverage plots were generated from the alignment les using the BEDTools genomecov function and displayed in the UCSC genome browser after conversion with the UCSC bedGraphToBigWig utility.
3
RNA-Seq Analysis of Hematopoietic Cells
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MOLM-13 cells or Lin-,cKit+ HSPCs were harvested and RNA was extracted using a quick-RNA Microprep kit (Zymo Research, CA). RNA-seq was done using TruSeq Stranded Total RNA library kit (Illumina, CA) as previously described [43 (link)]. The RNA-Seq paired-end reads were mapped to the mouse mm10 genome or human hg38 genome using STAR and quantified using RSEM [44 (link), 45 (link)]. Differentially expressed gene analysis and GSEA was done as previously described [42 (link), 46 (link)].
4
Next-Gen Sequencing of SARS-CoV-2 Spike Variant
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Total RNA was extracted from Vero CCL81 cells infected with VSV-SARS-CoV-2-SΔ21 using TRIzol (Invitrogen) according to the manufacturer’s protocol. RNA was used to generate next generation sequencing libraries using TruSeq Stranded Total RNA library kit with Ribo Zero ribosomal subtraction (Illumina). The libraries were quantified using a bioanalyzer (Agilent) and pooled at an equal molar concentration and used to generate paired end 250 bp reads on a MiSeq (Illumina). Raw sequencing data was processed using fastp v0.20.0 (Chen et al., 2018 (link)) to cut adaptor sequences and filter out sequences with a Phred quality score < 30. Processed reads were aligned to the reference plasmid sequence using BBMap v38.79 (Bushnell et al., 2017 (link)). Reads that mapped to the reference were extracted using SAMtools 1.9 (Li et al., 2009 (link)). The extracted mapped reads were used as input for de novo assembly with SPAdes v3.13.0 (Bankevich et al., 2012 ). Assembled contigs produced a 14.2 kb consensus sequence, which was subsequently aligned to the reference plasmid using NUCmer v3.1 (Delcher et al., 2002 (link)). Consensus sequences for each RNA sample were generated by aligning contigs to the reference plasmid sequence pVSV(1+)-eGFP-SARS-CoV-2-S with SnapGene v5.0.
5
RNA-seq Protocol for Tissue Transcriptome Analysis
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RNA was extracted from organs that were homogenized in Trizol LS using an Omni Tissue homogenizer. After incubation at RT for 5 min, samples were spun at 21 000 g for 3 min, supernatant transferred to a new tube and RNA extracted following manufacturer’s instructions. Strand-specific total RNA-seq libraries from ribosomal RNA-depleted RNA were prepared using the TruSeq Stranded Total RNA Library kit (Illumina) according to the manufacturer-supplied protocol. Libraries were sequenced 100 bp paired-end to a depth of 29.1–48.4 million reads on an Illumina HiSeq2500 instrument.
6
Comprehensive SARS-CoV-2 Genomic Profiling
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All 49 COVID-19 patients tested positive for SARS-CoV-2 by RT-qPCR using RNA extracted from nasopharyngeal swabs following the QIAamp Viral RNA Mini or the EZ1 DSP Virus Kits (Qiagen, Hilden, Germany). RNA libraries from all samples were then prepared for shotgun transcriptomic sequencing using the TruSeq Stranded Total RNA Library kit from Illumina (San Diego, CA, USA), following manufacturer’s instructions. Libraries were sequenced using the NovaSeq SP Reagent kit (2 X 150 cycles) from Illumina (San Diego, CA, USA). Sample L5630 underwent a target enrichment approach where double stranded DNA (synthesized using the QuantiTect Reverse Transcription Kit from Qiagen, Hilden, Germany) was amplified using 26 overlapping primer sets covering most of the SARS-CoV-2 genome as recently described by our group9 (link). PCR products were then sheared by ultra-sonication (Covaris LE220-plus series, MA, USA) and prepared for sequencing using the SureSelectXT Library Preparation kit (Agilent, CA, USA). This library was sequenced using the Illumina MiSeq Micro Reagent Kit, V2 (2 X 150 cycles).
7
RNA-Seq Library Preparation for Blood Samples
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For RNA-Seq library we used a TruSeq Stranded Total RNA library kit (Illumina), specifically tailored for blood samples, according to the manufacturer’s instructions. Briefly, 300 ng total RNAs were depleted in rRNA and globin mRNA (Ribo-Zero Globin), purified and fragmented into 250 bp fragments in average. First strand of cDNA was synthetized using Superscript II Reverse Transcriptase (Thermo Fisher Scientific, random primers) and the second strand cDNA using DNA Polymerase I and RNase H (UTP incorporation). DNA fragments were selectively enriched to obtain the library, then normalized, denatured (0.1 M NaOH) and sequenced (High-Output, 2 × 75 bp read length) on an Illumina NextSeq 500. Approximately 70 million reads per sample were acquired.
8
Small RNA and Bulk RNA Sequencing of Celiac Disease
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RNA was isolated from the small-intestinal biopsies using either the miRVana isolation Kit (Ambion, Carlsbad, CA, USA) or qiazol lysis reagent (Qiagen, Hilden, Germany, 79306). The proportion of CeD and controls did not differ between both isolation methods (χ2 P = 0.43). RNA quality was assessed using the Caliper GX bioanalyzer (Agilent, Santa Clara, CA, USA). Small RNA-libraries were generated starting from 500 ng total RNA using the TruSeq Small RNA Sample Prep kit (Illumina, San Diego, CA, USA), implementing 15 amplification cycles. RNA libraries were prepared as previously described in Zorro et al. [9 (link),62 (link)], using the Illumina TruSeq stranded total RNA library kit with a riboZero rRNA depletion step. After measurement of the cDNA concentration, libraries were pooled equimolarly per lane on a HiSeq 2500 (Illumina San Diego, CA, USA). Raw reads were aligned to miRbase 22 (small-RNA-seq) and human_g1k_v37 ensemble Release 75 (RNA-seq), as described previously (Tan et al. submitted manuscript; [62 (link)]). For all 43 samples, small-RNA libraries were generated and sequenced. Bulk RNA-sequencing was performed for 5/10 of the controls and 6/33 of the CeD patients (GEO accession number GSE146190) [9 (link),62 (link)].
9
Comprehensive Molecular Profiling of Metastatic Tumors
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All tumors were examined by a pathologist to determine tumor cellularity and necrosis and only samples of 60% tumor cellularity or higher with <20% necrosis were sequenced. DNA and RNA were extracted from FF or FFPE tissues using Qiagen’s DNeasy Blood & Tissue Kit and RNeasy kit. Whole-exome sequencing of DNA from matched primary tumor, metastatic tumors, and normal tissue samples was completed for 39 patients with the NimbleGen VCRome exome capture kit (NimbleGen Roche) according to the manufacturer’s protocol. Paired-end Illumina 151 bp reads were generated for normal samples to a minimum of depth of 65x, while tumor samples were sequenced to a minimum of 139x, with the average coverage of ~300x. A coverage table provides per-sample coverage details (Supplementary Table 1 ). RNA sequencing of primary and metastatic tumor samples was performed using the Illumina TruSeq stranded Total RNA library kit following the Manufacturer-recommended protocol. Paired-end Illumina sequencing of 151 bp read length yielded an average of approximately 125 million paired reads per-sample and an average of approximately 134 million reads mapped per sample. Quality Control metrics for the RNA-seq samples were generated using MultiQC and are reported in Supplementary Data 2 50 (link).
10
Bisulfite-seq library construction protocol
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The remaining RNA was combined into four final bsRNA-seq fractions (Fig. 1 ), and 10 μg RNA was spiked with ERCC Spike-in Mix 2 (Ambion) according to the manufacturer’s instructions. RNA was then used for sequencing library construction using the TruSeq® Stranded Total RNA Library Kit (Illumina) according to the manufacturer’s instruction, with some modifications. Following rRNA depletion, RNA was suspended in nuclease-free water and subjected to bisulfite conversion as described previously (T Sibbritt, A Shafik, SJ Clark and T Preiss) [86 (link)]. Converted RNA was purified, and 1 μg used for continued library preparation with omission of the fragmentation step as the RNA undergoes fragmentation during the bisulfite treatment. No size selection was performed as the size of the bisulfite treated RNA fragments was between 50 and 200 nt with a peak size of approximately 150 nt (Fig. S1 ). The libraries were mixed equally and loaded onto the HiSeq 2500 System (Illumina) using a total of three lanes for sequencing. Sequencing was performed in fast mode acquiring 101-bp paired-end reads.
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