Totalscript rna seq kit, by Illumina - Product details

1

RNA-Seq Analysis of Developmental Stages

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High-quality RNA (RIN>7) was extracted from all stages. Using the TotalScript RNA-Seq kit (Epicentre), two stranded libraries were prepared for each stage. This approach enabled low inputs (5ng of total RNA/reaction) and random hexamer priming to reduce polyA transcript bias. Each RNA pool was split once prior to adapter ligation and then split again prior to PCR amplification, resulting in four technical replicates per developmental stage. Purified libraries were quantified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay.
The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Library Quant Kit (Kapa Biosystems). Individual libraries were normalized to 10 nM and equal volumes were pooled in preparation for Illumina sequence analysis. Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq paired-end flow cell using an Illumina cBot. Flowcells were then transferred to an Illumina HiSeq 2000 instrument and sequenced in 100bp paired-end mode.

Hendrickson P.G., Doráis J.A., Grow E.J., Whiddon J.L., Lim J.W., Wike C.L., Weaver B.D., Pflueger C., Emery B.R., Wilcox A.L., Nix D.A., Peterson C.M., Tapscott S.J., Carrell D.T, & Cairns B.R. (2017). Conserved roles for murine DUX and human DUX4 in activating cleavage stage genes and MERVL/HERVL retrotransposons. Nature genetics, 49(6), 925-934.

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2

Bulk RNA-seq Protocol for Low-Input Samples

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3-10k cells were FACS sorted into buffer RLT with β-mercaptoethanol and total RNA prepared using RNAeasy Micro (Qiagen) with on column DNase I treatment. Stranded RNAseq libraries were prepared using the TotalScript RNAseq kit (Epicenter). Libraries were sequenced paired-end (2x50 cycles) on the Illumina platform (Illumina). Reads were mapped using STAR (v 2.5.2b) (32 (link)), strand-specific reads in exons quantified using HOMER (33 (link)) and significant changes identified using EdgeR (34 (link)). Only genes with ≥30 reads in at least two samples were considered in the analysis. Data was log transformed and quantile normalized for display. PCA and visualization were performed using R (v3.3.3). GO term analysis was done using Metascape (35 (link)) with standard settings.

Peña-Pérez L., Kharazi S., Frengen N., Krstic A., Bouderlique T., Hauenstein J., He M., Somuncular E., Li Wang X., Dahlberg C., Gustafsson C., Johansson A.S., Walfridsson J., Kadri N., Woll P., Kierczak M., Qian H., Westerberg L., Luc S, & Månsson R. (2022). FOXO Dictates Initiation of B Cell Development and Myeloid Restriction in Common Lymphoid Progenitors. Frontiers in Immunology, 13, 880668.

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3

RNA Expression Analysis in Mouse Cells

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RNA was extracted from cells or embryo tissues using the RNeasy Mini or Micro Kits (Qiagen). cDNA was synthesized from total RNA using the QuantiTect^® Reverse Transcription Kit (Qiagen). qPCR was performed using the GoTaq^® qPCR Master Mix (Promega) on the StepOnePlus Real-Time PCR System (Life Technologies). The following primer pairs were used: Ppp2r1a: fwd 5′-ccaccaagcacatgctgccc-3′, rev 5′-tccacatcctggtcctgggtca-3′; Pmm2: fwd 5′-agggaaaggcctcacgttct-3′; rev 5′-aataccgcttatcccatccttca-3′.
For RNA-seq the TotalScript™ RNA-Seq Kit (epicenter) was used for cDNA synthesis and library preparation. The sequencing was performed using the Illumina HiSeq 2500 (High Output) system for 50 bp paired end reads. Reads were mapped to the mouse genome mm10 (GRCm38) with TopHat (Trapnell et al., 2009 (link)) (v2.0.8b) and Cuffdiff (Trapnell et al., 2010 (link)) was used to calculate normalized FPKM values. Heat maps of differentially expressed genes and PCC were generated using the R program (http://www.r-project.org).

Lange L., Marks M., Liu J., Wittler L., Bauer H., Piehl S., Bläß G., Timmermann B, & Herrmann B.G. (2017). Patterning and gastrulation defects caused by the tw18 lethal are due to loss of Ppp2r1a. Biology Open, 6(6), 752-764.

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4

Single-Cell RNA-Seq Analysis of Cell Populations

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5-10 × 10³ cells were FACS-sorted into buffer RLT with β-mercaptoethanol and total RNA extracted using RNeasy Micro Kit (Qiagen, Hilden, Germany) with on-column DNase I treatment. Strand specific RNAseq libraries were prepared using the TotalScript™ RNA-seq kit (Epicenter, Madison, WI) together with custom made Tn5 (transposase). Barcoded libraries were pooled and pair-end sequenced (2 × 50 cycles) using the Illumina platform (NextSeq500, Illumina, San Diego, CA).
RNAseq reads were mapped using STAR (53 (link)), reads in exons quantified using HOMER (54 (link)) and significant changes identified using EdgeR. Principal component analysis (PCA) analysis and display was performed using R (v3.3.3). For details see Supplemental Materials and Methods.

Bouderlique T., Peña-Pérez L., Kharazi S., Hils M., Li X., Krstic A., De Paepe A., Schachtrup C., Gustafsson C., Holmberg D., Schachtrup K, & Månsson R. (2019). The Concerted Action of E2-2 and HEB Is Critical for Early Lymphoid Specification. Frontiers in Immunology, 10, 455.

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5

Genomic and Transcriptomic Profiling of Brain

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A short-insert and two mate-pair libraries were prepared from the brain genomic DNA of an individual using TruSeq DNA sample preparation kit v2 (Illumina, San Diego, USA) and Nextera mate-pair library preparation kit (Illumina), respectively. Mate-pair libraries were prepared from two size ranges of tagmented DNA fragments, 4 kb and 8 kb in average. RNA-seq libraries were prepared from the total RNA samples of the same individual (brain and arm tips) using TotalScript RNA-Seq Kit (Epicentre, Wisconsin, USA). The DNA- and RNA-seq libraries were sequenced on HiSeq1500/2500 systems (Illumina). Paired-end read (150 bp × 2) sequencing was performed using the HiSeq Rapid SBS Kit HS. The conversion of bcl files to fastq files was performed using the CASAVA v1.8.2 (configureBclToFastq.pl) software (Illumina).

Yoshida M.A., Imoto J., Kawai Y., Funahashi S., Minei R., Akizuki Y., Ogura A., Nakabayashi K., Yura K, & Ikeo K. (2020). Genomic and Transcriptomic Analyses of Bioluminescence Genes in the Enope Squid Watasenia scintillans. Marine Biotechnology (New York, N.y.), 22(6), 760-771.

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6

RNA-Seq Analysis of Developmental Stages

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High-quality RNA (RIN>7) was extracted from all stages. Using the TotalScript RNA-Seq kit (Epicentre), two stranded libraries were prepared for each stage. This approach enabled low inputs (5ng of total RNA/reaction) and random hexamer priming to reduce polyA transcript bias. Each RNA pool was split once prior to adapter ligation and then split again prior to PCR amplification, resulting in four technical replicates per developmental stage. Purified libraries were quantified on an Agilent Technologies 2200 TapeStation using a D1000 ScreenTape assay.
The molarity of adapter-modified molecules was defined by quantitative PCR using the Kapa Library Quant Kit (Kapa Biosystems). Individual libraries were normalized to 10 nM and equal volumes were pooled in preparation for Illumina sequence analysis. Sequencing libraries (25 pM) were chemically denatured and applied to an Illumina HiSeq paired-end flow cell using an Illumina cBot. Flowcells were then transferred to an Illumina HiSeq 2000 instrument and sequenced in 100bp paired-end mode.

Hendrickson P.G., Doráis J.A., Grow E.J., Whiddon J.L., Lim J.W., Wike C.L., Weaver B.D., Pflueger C., Emery B.R., Wilcox A.L., Nix D.A., Peterson C.M., Tapscott S.J., Carrell D.T, & Cairns B.R. (2017). Conserved roles for murine DUX and human DUX4 in activating cleavage stage genes and MERVL/HERVL retrotransposons. Nature genetics, 49(6), 925-934.

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7

Transcriptome Profiling of LSK-SLAM Cells

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RNA was isolated from 15,000 LSK-SLAM cells (Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺) using the Nucleospin XS Kit (Macherey Nagel) and quantified on Bionalyzer using RNA Pico 6000 Kit (Agilent). Ribosomal depletion was performed using a modified version of RiboZero Kit (Illumina). In all, 300 pg ribosomal-depleted RNA was used as input into TotalScript RNA-Seq Kit (Epicentre). Libraries were pooled and sequenced to 30–50 million reads on HiSeq 2500.

Renders S., Svendsen A.F., Panten J., Rama N., Maryanovich M., Sommerkamp P., Ladel L., Redavid A.R., Gibert B., Lazare S., Ducarouge B., Schönberger K., Narr A., Tourbez M., Dethmers-Ausema B., Zwart E., Hotz-Wagenblatt A., Zhang D., Korn C., Zeisberger P., Przybylla A., Sohn M., Mendez-Ferrer S., Heikenwälder M., Brune M., Klimmeck D., Bystrykh L., Frenette P.S., Mehlen P., de Haan G., Cabezas-Wallscheid N, & Trumpp A. (2021). Niche derived netrin-1 regulates hematopoietic stem cell dormancy via its receptor neogenin-1. Nature Communications, 12, 608.

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8

FACS-Purified Macrophage RNA Isolation and Sequencing

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RNA isolation from FACS-purified macrophages was done using the QIAgen Rneasy Micro Kit and QIAgen Rneasy Micro Kit where cell number was less than 5×10⁵ cells. RNA-Seq library preparation was done at the NYU School of Medicine Genome Technology Core using a low-input protocol with the TotalScript RNA-Seq kit (Epicentre). Libraries were sequenced on the HiSeq 2000 (Illumina) with 2 × 50 cycles and for an average of 50 million reads per sample.

Gundra U.M., Girgis N.M., Gonzalez M.A., Tang M.S., Van Der Zande H.J., Lin J.D., Ouimet M., Ma L.J., Poles J.A., Vozhilla N., Fisher E.A., Moore K.J, & Loke P. (2017). Vitamin A mediates conversion of monocyte-derived macrophages into tissue resident macrophages during alternative activation. Nature immunology, 18(6), 642-653.

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9

FACS-Purified Macrophage RNA Isolation and Sequencing

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RNA isolation from FACS-purified macrophages was done using the QIAgen Rneasy Micro Kit and QIAgen Rneasy Micro Kit where cell number was less than 5×10⁵ cells. RNA-Seq library preparation was done at the NYU School of Medicine Genome Technology Core using a low-input protocol with the TotalScript RNA-Seq kit (Epicentre). Libraries were sequenced on the HiSeq 2000 (Illumina) with 2 × 50 cycles and for an average of 50 million reads per sample.

Gundra U.M., Girgis N.M., Gonzalez M.A., Tang M.S., Van Der Zande H.J., Lin J.D., Ouimet M., Ma L.J., Poles J.A., Vozhilla N., Fisher E.A., Moore K.J, & Loke P. (2017). Vitamin A mediates conversion of monocyte-derived macrophages into tissue resident macrophages during alternative activation. Nature immunology, 18(6), 642-653.

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10

RNA-Seq Library Preparation and Sequencing

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RNA was quantified and treated with DNase according to the manufacturers protocol (Qiagen, Inc). RNA was used for generating sequencing libraries using the TotalScript™ RNA-Seq Kit (Epicentre), providing total RNA for sequencing without normalization. The libraries were sequenced at the Center for Cancer Research Sequencing facility using the HiSeq 2000 (Illumina) with paired end 100bp reads using the TruSeq Cluster Kit v3 (Illumina). Data was converted to fastq and aligned back to the human genome assembly 19 with the STAR split-read aligner. RNAeQC was used to analyze data quality (23 (link)). The Binary Sequence Alignment Map (BAM) files are deposited and available from the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA433861).

Reinhold W.C., Varma S., Sunshine M., Elloumi F., Ofori-Atta K., Lee S., Trepel J.B., Meltzer P.S., Doroshow J.H, & Pommier Y. (2019). RNA sequencing of the NCI-60: Integration into CellMiner and CellMiner CDB. Cancer research, 79(13), 3514-3524.

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Totalscript rna seq kit

Lab products found in correlation

10 protocols using totalscript rna seq kit

RNA-Seq Analysis of Developmental Stages

Bulk RNA-seq Protocol for Low-Input Samples

RNA Expression Analysis in Mouse Cells

Single-Cell RNA-Seq Analysis of Cell Populations

Genomic and Transcriptomic Profiling of Brain

RNA-Seq Analysis of Developmental Stages

Transcriptome Profiling of LSK-SLAM Cells

FACS-Purified Macrophage RNA Isolation and Sequencing

FACS-Purified Macrophage RNA Isolation and Sequencing

RNA-Seq Library Preparation and Sequencing

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