RNA isolation was performed following RNAqueous Micro kit (Life Technologies) directions, including optional DNAse treatment. SoLo RNA-Seq System (NuGEN) was used to construct cDNA libraries, following manufacturer’s directions. Libraries were screened for quality on a Bioanalyzer (Agilent). No more than 50% of the NuGEN libraries were pooled with other TruSeq libraries to maintain the sequence diversity. Using TruSeq SR Cluster Kit v3, at 15 pM the libraries were clustered onto the flow cell and sequenced on HiSeq 1000 (Illumina) using Illumina standard sequencing primer. At single read 1× 51 bp setting, at least 45 million reads per sample were generated.
Truseq sr cluster kit
Manufactured by Illumina
Sourced in United States
The TruSeq SR Cluster Kit is a laboratory equipment product used for preparing single-read sequencing libraries. It is designed to facilitate the generation of clusters for Illumina sequencing platforms.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (5 mentions)
Hiseq 2000, by Illumina (4 mentions)
Kapa stranded rna seq library prep kit, by Illumina (4 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (3 mentions)
Truseq sbs kit v3 hs reagents, by Illumina (3 mentions)
Truseq stranded mrna library preparation kit, by Illumina (3 mentions)
Truseq v3 flowcell, by Illumina (3 mentions)
D1000 screentape assay, by Agilent Technologies (3 mentions)
Tapestation 2200, by Agilent Technologies (3 mentions)
Ribo zero magnetic gold kit, by Illumina (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Truseq sbs kit, by Illumina (2 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (2 mentions)
Hiseq 4000 sequencing platform, by Illumina (2 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (2 mentions)
Truseq rna sample preparation v2 guide, by Illumina (2 mentions)
Truseq sbs v3 reagents, by Illumina (2 mentions)
Rnaqueous micro kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 1000, by Illumina (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
17 protocols using truseq sr cluster kit
1
Robust RNA-Seq Library Preparation
2
Transcriptional Profiling of p73 in MTECs
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Gene expression for wild-type and p73 knockout MTECs at ALI D0, D4, D7, and D14 was measured. RNA was isolated with RNeasy kit (Qiagen). RNA-seq and sRNA-seq libraries were prepared by TruSeq kits (Illumina). Library quality and sample concentration were checked before sequencing (50-base-pair single end) on a HiSeq 2000 (Illumina) using TruSeq SR cluster kit version 3-cBot-HS and TruSeq SBS kit version 3-HS. All experiments ran in triplicate, sequencing three biological replicates per condition. Murine RNA-seq data were aligned to the Mus musculus mm10 genome using STAR aligner (version 2.3.0e_r291) with default options. Read counts for all genes and all exons (Ensembl annotation version 72) were obtained using FeaturesCount (version 1.4.6) (Liao et al. 2014 (link)). To identify enriched GO categories, the Web service WebGestalt was used. GO category enrichment was assessed by calculating the fold change between observed and expected numbers of genes of a given GO category, where terms were scored enriched if they had an adjusted P-value of <0.1 (GEO no. GSE75717).
3
Plasma RNA Sequencing Protocol
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Total RNA was extracted from plasma samples using TRIzol LS Reagent (Invitrogen), according to the manufacturer’s instructions. Sequencing libraries were generated using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs), RiboZero Magnetic Gold Kit (Epicenter, Illumina Company), and KAPA Stranded RNA-Seq Library Prep Kit (Illumina). An Agilent Bioanalyzer 2100 system (Agilent) was used to qualify the sequencing libraries. Finally, high-throughput NGS was carried out using the TruSeq SR Cluster Kit (Illumina), based on the Illumina HiSeq 4000 sequencing platform (Illumina). The sequencing data has been uploaded to ArrayExpress database (E-MTAB-10494) .
4
TruSeq DNA Library Preparation Protocol
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The TruSeq DNA Sample Prep Kit v2 (Illumina) was used in the succeeding steps. DNA samples (1 μg) were sonicated and the fragmented DNA samples end-repaired and polyadenylated. TruSeq adapters containing the index for multiplexing were ligated to the fragmented DNA samples. The ligated samples were run on a 2% agarose gel and the desired fragment length was excised (50bp +/- the target fragment length). DNA from the gel was purified with MinElute Gel Extraction Kit (Qiagen). Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using Qubit (1.0) Fluorometer and the Caliper GX LabChip GX (Caliper Life Sciences). The product is a smear with an average fragment size of approximately 260 bp. The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit (Illumina) was used for cluster generation using 5 pM of pooled normalized libraries on the cBOT. Sequencing of single reads was performed on one lane of the Illumina HiSeq 2000 using the TruSeq SBS Kit v3-HS (Illumina Inc, USA).
5
Transcriptome Analysis of Rice Tissues
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The pooled RNA samples extracted from the leaves, flowers and stems of rice obtained during the blooming stage were sent for sequencing services by the Kangchen Bio-tech Inc., China. The quality and quantity of RNA samples were analyzed by NanoDrop ND-1000 model. The quantified RNA was used to synthesize cDNA, followed by the ligation of 5’- and 3’-adapters to create the sequencing library. The cDNA samples were diluted to a final concentration of 8 pM prior to generate clusters on Illumina cBlot using TruSeq SR Cluster kit (#GD-402-4001, Illu-mina). Sequencing was performed on Illimina HiSeq 2000 using TruSeq Rapid SBS Kit (#FC-402-4001, Illumina), followed by data acquisition and processing sequence. The sequence data have been deposited in the NCBI GEO under accession number GSE224933.
6
RNA-seq of TRPV1 Variants in NHBE Cells
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RNA sequencing was performed as previously described15 (link) at the High Throughput Genomics Core Facility at the Huntsman Cancer Institute, University of Utah. NHBE cells from the four donors (14359 and 14664 for TRPV1 I585I/I, and 9853 and unknown for TRPV1 I585I/V) were used. Total RNA was extracted using the RNeasy Mini kit (Qiagen) with on-column DNase digestion. RNA quality was assessed by RNA nanochip technology, and library construction was performed using the Illumina TruSeq Stranded mRNA Sample Preparation kit using established protocols. The sequencing libraries (18 pM) were then chemically denatured and applied to an Illumina TruSeq version 3 single-read flow cell using an Illumina cBot. Hybridized molecules were clonally amplified and annealed to sequencing primers with reagents from an Illumina TruSeq SR Cluster kit, version 3-cBot-HS. Following transfer of the flow cell to an Illumina HiSeq instrument, a 50-cycle single-read sequence run was performed using TruSeq SBS version 3 sequencing reagents. Data were processed at the University of Utah Bioinformatics core. The data presented in this publication have been deposited in the National Center for Biotechnology Information’s (NCBI’s) Gene Expression Omnibus (GEO) and are accessible through GEO Series accession no. GSE85447 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE85447 ).
7
RNA-Seq Library Preparation and Sequencing
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Libraries were constructed using 1μg total RNA, following the Illumina TruSeq RNA Sample Preparation v2 Guide. The products were amplified by PCR to create a cDNA library, which was then validated on the Agilent 2100 Bioanalyzer using DNA-1000 chip. Cluster generation was performed on the Illumina cBot, using a Single Read Flow Cell with a Single Read cBot reagent plate (TruSeq SR Cluster Kit). Sequencing of the clustered flow cell was performed on the Illumina HiSeq 2000, using TruSeq SBS v3 reagents (Illumina, San Diego, CA).
8
MSC Total RNA Sequencing Pipeline
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Total RNA was extracted from MSCs using RNAiso (Takara Bio, Shiga, Japan), and reverse transcription was performed using RNA PCR Kit (Takara Bio, Shiga, Japan). Agarose gel electrophoresis was used for integrity and quality testing of total RNA. Then, 1–2 μg of total RNA was used to construct sequencing libraries using the KAPA Stranded RNA-Seq Library Prep Kit (Illumina, Hayward, CA, USA) according to the manufacturer’s instructions. The mixed sample sequencing library was transformed by NaOH to generate single-stranded DNA and diluted to a concentration of 8 pM and then, amplified by the TruSeq SR Cluster Kit (Illumina, Hayward, CA, USA). The ends of the generated fragments were sequenced 150 cycles using a HiSeq 4000 device (Illumina, Hayward, CA, USA).
9
Single-Strand DNA Generation and Sequencing
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The mixed sample sequencing library was transformed by NaOH to generate single-stranded DNA and diluted to a concentration of 8 pM. Then amplified by the TruSeq SR Cluster Kit (Illumina, America), and the ends of the generated fragments were sequenced 150 cycles through Illumina HiSeq 4000 (Illumina, America).
10
RNA-Seq Library Preparation and Sequencing
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