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Mgieasy rna directional library prep kit

Manufactured by MGI Tech
Sourced in China

The MGIEasy RNA Directional Library Prep Kit is a laboratory equipment product designed for the preparation of RNA libraries. The kit provides the necessary reagents and protocols to facilitate the construction of directional RNA libraries, which are used in various genomic analysis and sequencing applications.

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7 protocols using mgieasy rna directional library prep kit

1

Whole-Transcriptome Sequencing of PtNP-Treated THP-1 Cells

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THP-1 cells were incubated with IC50 concentrations of PtNPs for 24 h. Total RNA was isolated from control or treated cells using TRI reagent (Merck, Darmstadt, Germany). In total, 500 ng of total RNA was processed for the preparation of the whole-transcriptome sequencing library. Depletion of ribosomal RNA (rRNA) was performed using an MGIEasy RNA Directional Library Prep Kit (MGI Tech, Shenzhen, China) according to the manufacturer’s instruction. The total RNAs were fragmented and copied into first-strand complementary DNA (cDNA) using reverse transcriptase and random primers. Strand specificity was achieved in the RT directional buffer, followed by second-strand cDNA synthesis. Subsequently, the cDNA fragments were ligated to sequencing adapter. The products were then purified and enriched with PCR amplification. The double-stranded library was quantified using a QauntiFluor ONE dsDNA System (Promega, Madison, WI, USA). The library was cyclized at 37 °C for 60 min, and digested at 37 °C for 30 min, followed by cleanup of the circularization product. To make a DNA nanoball (DNB), the library was incubated at 30 °C for 25 min using a DNB enzyme. Finally, the library was quantified by a QauntiFluor single-stranded (ss)DNA System (Promega, Madison, WI, USA) and sequenced on the MGIseq system (MGI Tech, Shenzhen, China) with 100-bp paired-end reads.
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2

Transcriptome Analysis of MEIS1 Knockdown

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GSC11 (3.5×105 cells/well) was seeded in a 6-well plate (laminin-coated). Cells were transfected with either shNT control or shMEIS1_72 lentivirus and then incubated for 48 h. At the end of the incubation period, cells were resuspended in TRIzol® Reagent (Invitrogen; CA, USA) and kept at −80°C. Libraries were constructed using the MGIEasy RNA Directional Library Prep Kit (MGI Tech., Shenzhen, China) following the manufacturer’s procedure. RNA quality control and high-throughput RNA sequencing were performed on the MGISEQ platform (MGI Tech) with 149 bp paired-end reads. Reads were mapped to the hg19 reference genome and expression values were calculated in fragments per kb transcript per million fragments mapped (FPKM). LAS Co., Ltd. (Gimpo, South Korea) performed the RNA purification, library preparation, and sequencing procedures. The new generation of high-throughput sequencing was used to study the differences between the two samples and gene expression in both groups was compared using EdgeR. Significant genes (Log2(FC) cut-off >0.5 and <0.5; p ≤ 0.05) were used for functional annotation on the DAVID website. GSEA analysis was also performed to identify the downregulated gene sets after MEIS1 knockdown.
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3

Transcriptomics Analysis of iPSC Response to ECR

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Total RNAs were isolated from iPSCs treated with 5 μg/mL and 10 μg/mL ECR using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and RNA quality was assessed by 2100 Bioanalyzer Instrument (Agilent, Santa Clara, CA, USA). The library was prepared using the MGIEasy RNA Directional Library Prep Kit and sequenced using MGISEQ-2000 (MGI Tech, Shenzhen, China) to generate 100-bp paired-end reads. Reads were trimmed using Trim Galore to remove adapter sequences and reads with low sequence quality. High-quality sequence reads were mapped to the human genome (hg38), and the expression levels of mRNAs were quantified using the DESeq2 [19 (link)]. The differences in expression levels between ECR treatment and control groups, in terms of the rate of change (log transformation) and statistical significance (false discovery rate; FDR < 0.01), were analyzed using the edgeR package [20 (link)] in R. ClueGO [21 (link)], a Cytoscape plug-in tool, was used to functionally grouped gene ontology (GO) and pathway annotation networks.
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4

mRNA Sequencing Library Preparation

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Total RNA (1 µg) was processed to prepare the mRNA sequencing library following the manufacturer’s instructions provided with the MGIEasy RNA Directional Library Prep kit (#1000006386; MGI Tech, Shenzhen, China). The constructed library was quantified using a QauntiFluor® ssDNA System (E3190; Promega). Subsequently, the prepared DNA nanoballs were sequenced on the MGISeq platform (MGI Tech) with 100 bp paired-end reads. FastQC (v0.11.9) was used to assess the read quality. Common sections of the MGISEQ adapter sequences were eliminated using TrimGalore (v0.6.5). The resulting trimmed reads were mapped to the GRCm38 (mm10) mouse reference genome using STAR (v2.7.3a) [24 (link)] with default configurations. To quantify gene expression levels, we used RSEM (v1.3.3) [25 (link)] along with the GRCm38.86 gene annotation to obtain the expected read counts and transcript per million (TPM) values.
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5

RNA-seq Analysis of Human Herpesvirus 1

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RNA sequencing was performed in collaboration with BGI Europe Genome Center (Copenhagen, Denmark) following the standard operational procedure as described before42 . Briefly, the quality of total RNA was checked using the Agilent 2100 bioanalyzer. To construct the sequencing library for MGIseq-2000, ~1 μg of polyA enriched RNA was used for library construction using the MGIEasy RNA Directional Library Prep Kit (MGI Tech). Next, paired-end sequencing with 100 cycles was performed using the MGISEQ-2000 sequencing instrument, according to the manufacturer’s instructions. We generated an average of 63 million raw reads for each sample. The clean RNA reads were first aligned to the hg19 UCSC RefSeq (RNA sequences, GRCh37) using bowtie2 at first. To map the transcripts from the viruses, the unmapped reads were then aligned to the coding sequence of the human herpesvirus 1 (KOS strain). The expression of human genes and virus genes were performed by transforming mapped transcript reads to TPM using RSEM43 . The normalized expression were estimated and normalized by DEseq2. Differentially expressed genes were defined as genes with fold change over twofolds and adjusted p value < 0.001 using DESeq2.
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6

Whole Transcriptome Sequencing Library Preparation

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The value of RNA Integrity Number (RIN) and the DV200 metric were measured using an RNA 6000 Nano Kit and an Agilent 2100 Bioanalyzer (Agilent Technologies) to confirm the quality and integrity of the RNA. RNA samples with a RIN >7 were designated as “good total RNA quality” and were selected for downstream applications. Total RNA (500 ng) was processed to prepare a whole transcriptome sequencing library. Enrichment of whole transcriptome RNA was conducted by depleting ribosomal RNA (rRNA) prior to preparation of the whole transcriptome sequencing library using the MGIEasy RNA Directional Library Prep Kit (MGI Tech Co., Ltd.) as described previously [16 (link)]. Reference genome sequence data from Homo sapiens were obtained from the NCBI Genome database (assembly ID: GRCh38). Reference genome indexing and read mapping of tissue samples were performed using STAR software (ver. 2.5.4b).
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7

RNA Sequencing Library Preparation

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All RNA samples were used for library preparation using the MGIEasy RNA Directional Library Prep Kit (MGI Tech., China). RNA quality control and RNA sequencing were performed by LAS Inc. (Korea) on the Illumina MGISEQ platform (Illumina Inc., USA). RNA quality was measured by the absorbance at 260, 280, and 230 nm (LAS Inc.).
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