Gene expression sample preparation kit

Manufactured by Illumina

Sourced in United States, China

The Gene Expression Sample Preparation Kit is a laboratory equipment product designed for the preparation of samples for gene expression analysis. It provides the necessary components and protocols to extract, purify, and process RNA samples in a streamlined manner. The kit is intended to facilitate the initial steps of the gene expression workflow, enabling researchers to proceed with downstream analysis.

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11 protocols using gene expression sample preparation kit

RNA Extraction and Sequencing from Plant Tissues

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Total RNA was extracted from root or leaf samples using the CTAB method [49 (link)]. The isolated RNA was subsequently treated with RNase-Free DNase I (Roche, http://www.roche.com).
Two biological replicates from leaves and roots sampled at each of the three time points, from both drought-stressed and control plants, were sequenced, making a total of 24 sequencing samples. Tag libraries from the RNA samples were prepared in parallel using an Illumina gene expression sample preparation kit and sequenced using the Illumina GAII platform at BGI-Shenzhen (http://en.genomics.cn/navigation/index.action) [50 (link)]. For gene expression analysis, the number of expressed tags was calculated and then normalized to TPM (number of transcripts per million tags) [51 (link)]. The sequencing saturation statistics are shown in Additional file 1: Table S1.

Zhang C., Zhang L., Zhang S., Zhu S., Wu P., Chen Y., Li M., Jiang H, & Wu G. (2015). Global analysis of gene expression profiles in physic nut (Jatropha curcas L.) seedlings exposed to drought stress. BMC Plant Biology, 15, 17.

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Illumina-based Transcriptome Profiling

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Next‐generation sequencing analyses were performed for two biological replicates on an Illumina Cluster Station and the Illumina HiSeq 2000 System using primarily reagents from the Illumina Gene Expression Sample Preparation Kit and the Illumina Sequencing Chip (Flowcell; Illumina, San Diego, CA, USA). Sequence tags were prepared using the Digital Gene Expression Tag Profiling Kit (Illumina), according to the Illumina protocol.

Mavromatis C.(., Bokil N.J., Totsika M., Kakkanat A., Schaale K., Cannistraci C.V., Ryu T., Beatson S.A., Ulett G.C., Schembri M.A., Sweet M.J, & Ravasi T. (2015). The co‐transcriptome of uropathogenic E scherichia coli‐infected mouse macrophages reveals new insights into host–pathogen interactions. Cellular Microbiology, 17(5), 730-746.

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Transcriptomics of Bamboo Flowering Stages

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Tag library preparation for samples of the four different flowering developmental periods (F1, F2, F3 and F4) was performed in parallel using the Illumina gene expression sample preparation kit. Transcriptome data of nonflowering moso bamboo leaves (CK) were regarded as the reference gene database, and the four libraries were sequenced using the Illumina high‐seq 2000 at Beijing Genomics Institute (BGI) (Shenzhen, Guangdong Province, China) (Gao et al., 2014).
Total RNA was extracted from the frozen samples (CK, F1, F2, F3 and F4) using the Trizol reagent (Invitrogen, Carlsbad, California, USA), according to the manufacturer's instructions. Five small RNA libraries were constructed for moso bamboo, and the Illumina high‐seq 2000 sequencing was carried out by the Beijing Genomics Institute (BGI) (Shenzhen, Guangdong Province, China) (Gao et al., 2015).
KEGG pathway analysis was performed after we obtained the sequencing data. Key regulation pathways and genes were selected according to expression abundance. Hierarchical clustering of expressional data was carried out using the Cluster 3.0 and Treeview programs (Eisen et al., 1998).

Ge W., Zhang Y., Cheng Z., Hou D., Li X, & Gao J. (2016). Main regulatory pathways, key genes and microRNAs involved in flower formation and development of moso bamboo (Phyllostachys edulis). Plant Biotechnology Journal, 15(1), 82-96.

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Profiling Renal lncRNAs, miRNAs, and mRNAs

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The lncRNA, microRNA and mRNA profiles in rat kidney tissues were assessed by next-generation sequencing. rRNA-depleted total RNA was purified from 6 kidney tissues from PC-AKI and control rats using a Ribo-Zero rRNA Removal Kit (Epicentre). Libraries were constructed using an Illumina Gene Expression Sample Preparation Kit and sequenced on an Illumina HiSeq^TM 2000 system (Beijing Genomics Institute (BGI)) with 50-60 million 2×100 bp paired raw passing filter reads. The raw reads were filtered to obtain the high-quality reads by removing reads containing an adaptor sequence or reads with a percentage of unknown bases (N) exceeding 10%; low-quality reads contained more than 50% unknown bases and had a Q value of <5. The high-quality reads were mapped to the rat reference genome (IRGSP build 5.0) using SOAPaligner/SOAP2, allowing no more than two mismatches in the alignment. Manipulation of the false discovery rate (FDR) was used to determine the threshold P values in multiple testing and analyses.

Chen G., Liu B., Chen S., Li H., Liu J., Mai Z., Chen E., Zhou C., Sun G., Guo Z., Lei L., Huang S., Zhang L., Li M., Tan N., Li H., Liao Y., Liu J., Chen J, & Liu Y. (2021). Novel biomarkers for post-contrast acute kidney injury identified from long non-coding RNA expression profiles. International Journal of Biological Sciences, 17(3), 882-896.

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Digital Gene Expression Library Construction

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Digital gene expression (DGE) libraries were constructed using an Illumina Gene Expression Sample Preparation Kit according to the manufacturer’s instructions. A total of 24 libraries derived from immature fibers at 5, 10, 15 and 20 DPA were constructed and sequenced using the Solexa Genome Sequencing Analyzer system provided by BGI (Beijing Genomics Institute at Shenzhen, China), which was described in detail previously [39 (link)].

Fang L., Tian R., Li X., Chen J., Wang S., Wang P, & Zhang T. (2014). Cotton fiber elongation network revealed by expression profiling of longer fiber lines introgressed with different Gossypium barbadense chromosome segments. BMC Genomics, 15(1), 838.

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Tissue-Specific Transcriptome Profiling

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Total RNA from leaf, fruit, petal, sepal, ovary, stem and bud was extracted using an RNAprep Pure Plant Kit (Polysaccharides & Polyphenolics-rich) (Tiangen, Beijing, China) following the manufacturer’s instructions and dissolved in RNase-free DNase I (Thermo, USA) to remove residual DNA. Total RNA was treated using oligo(dT) magnetic beads to purify mRNA, which was then fragmented using sonication. First-strand and second-strand cDNA was synthesized using random hexamer primers, and double-stranded cDNA was ligated to an A-tail and special sequencing adaptor (Illumina gene expression sample preparation kit, San Diego, CA). PCR was performed with Phusion High-Fidelity DNA polymerase, universal PCR primers and Index (X) Primer. The library preparations were sequenced on an Illumina HiSeq platform to generate 125 bp/150 bp paired-end reads (Novogene, Beijing, China). Leaves and fruits were replicated two times, and the other tissues were replicated three times.

Li Q., Qiao X., Yin H., Zhou Y., Dong H., Qi K., Li L, & Zhang S. (2019). Unbiased subgenome evolution following a recent whole-genome duplication in pear (Pyrus bretschneideri Rehd.). Horticulture Research, 6, 34.

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RNA-Seq of Dental Pulp Stem Cells

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We used DPSCs for RNA sequencing. We constructed libraries using the Illumina Gene Expression Sample Preparation Kit and sequenced them with the Illumina HiSeqTM 2000 (next generation sequencing) from Beijing Genomics Institute, Beijing, China. In brief, we isolated total RNA from each sample and treated them with DNase I to degrade any potential DNA contamination, then enriched mRNA with oligo (dT) magnetic beads. We mixed enriched mRNA with fragmentation buffer and fragmented it into short fragments (∼200 bp) from which we synthesized the first strand via a random hexamer primer. We synthesized the second strand after adding reaction buffer, RNaseH, dNTPs, and DNA polymerase I to the first strand synthesis mixture. We purified double-stranded cDNA with magnetic beads and added a 3′-terminal single nucleotide adenine. Lastly, we ligated sequencing adaptor to the fragment to amplify it by PCR. We sequenced enriched fragments by the Illumina HiSeq^TM 2000 and generated 50-bp raw reads by the Illumina Genome Analyzer II.

Ji F., Zhu L., Pan J., Shen Z., Yang Z., Wang J., Bai X., Lin Y, & Tao J. (2020). hsa_circ_0026827 Promotes Osteoblast Differentiation of Human Dental Pulp Stem Cells Through the Beclin1 and RUNX1 Signaling Pathways by Sponging miR-188-3p. Frontiers in Cell and Developmental Biology, 8, 470.

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Leaf Transcriptome Analysis Pipeline

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Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), after which the concentration, quality, and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Wilmington, DE, USA). Twelve independent leaf complementary DNA (cDNA) libraries which stand for four different experimental conditions with three biological repetitions were constructed using the Illumina Gene Expression Sample Preparation Kit according to the manufacturer’s instructions, and 125 paired end reads were sequenced at the Norwegian Sequencing Centre. All the sequenced data is available at National Center for Biotechnology Information (NCBI) under the bioproject accession number PRJNA602126.

Su H., Zhang X., He Y., Li L., Wang Y., Hong G, & Xu P. (2020). Transcriptomic Analysis Reveals the Molecular Adaptation of Three Major Secondary Metabolic Pathways to Multiple Macronutrient Starvation in Tea (Camellia sinensis). Genes, 11(3), 241.

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Constructing Digital Gene Expression Libraries

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Digital gene expression libraries were constructed using the Illumina Gene Expression Sample Preparation Kit according to the manufacturer's instructions. We constructed and sequenced 14 libraries derived from immature fibers at 15, 20, and 25 DPA using the Solexa Genome Sequencing Analyzer system provided by BGI (Beijing Genomics Institute at Shenzhen, China), which gave 21 bp tags. The process was described in detail previously [18] (link).

Fang L., Tian R., Chen J., Wang S., Li X., Wang P, & Zhang T. (2014). Transcriptomic Analysis of Fiber Strength in Upland Cotton Chromosome Introgression Lines Carrying Different Gossypium barbadense Chromosomal Segments. PLoS ONE, 9(4), e94642.

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Sequencing Floral Developmental Stages

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Tag library preparation for samples of four different flowering developmental periods (floral bud formation, inflorescence development, anthesis and embryo formation stages) was performed in parallel using the Illumina gene expression sample preparation kit. Briefly, mRNA was extracted using magnetic oligo (dT) beads from total RNA. Purified mRNA was then mixed with the fragmentation buffer and fragmented into short fragments, which were used as templates for cDNA synthesis. Purified short cDNA fragments were resolved in EB buffer for end reparation and single nucleotide A (adenine) addition, and then they were connected with adapters. Subsequently, suitable cDNA fragments were selected for the PCR amplification as templates. During the QC steps, quantification and qualification of the sample library were assessed using an Agilent 2100 Bioanaylzer and an ABI StepOnePlus Real-Time PCR System. Finally, the library was sequenced using Illumina HiSeq 2000 at Beijing Genomics Institute (BGI) in Shenzhen, China.

Gao J., Zhang Y., Zhang C., Qi F., Li X., Mu S, & Peng Z. (2014). Characterization of the Floral Transcriptome of Moso Bamboo (Phyllostachys edulis) at Different Flowering Developmental Stages by Transcriptome Sequencing and RNA-Seq Analysis. PLoS ONE, 9(6), e98910.

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