Total RNA (1 µg) was processed to prepare the mRNA sequencing library using the MGIEasy RNA Directional Library Prep kit (MGI Tech Co., Ltd., China) according to the manufacturer’s instructions. The library was quantified using the QauntiFluor® ssDNA System (Promega Corporation, WI, United States). The prepared DNA nanoball was sequenced on the MGIseq system (MGI Tech Co., Ltd., China) with 100 bp paired-end reads. RNA-seq raw data quality was checked using FastQC (v0.11.9). TrimGalore (v0.6.5) was used to remove the common regions of the MGISEQ adapter sequences. The cleaned reads were mapped to the human genome assembly GRCh38 (hg38) using the STAR aligner (v2.7.3a) with default settings. Transcript abundance per gene, such as expected read count or transcripts per million, was quantified by RSEM (v1.3.3) with human gene annotation GRCh38.84. The raw sequence data (FASTQ files) and pre-processed count data were deposited in the Gene Expression Omnibus with accession number GSE182007. Differential gene expression analysis between the groups (e.g., BR treatment vs. vehicle) was conducted using the edgeR package (v3.38) in R (v3.6.3), yielding a ranked list of genes based on log2FC and adjusted p-value.
Qauntifluor ssdna system
Manufactured by Promega
Sourced in United States
The QuantiFluor ssDNA System is a fluorometric assay that provides a sensitive and specific method for the quantification of single-stranded DNA (ssDNA) in solution. The system utilizes a proprietary fluorescent dye that selectively binds to ssDNA, allowing for accurate measurement of ssDNA concentration.
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Lab products found in correlation
5 protocols using qauntifluor ssdna system
1
RNA-seq Library Preparation and Analysis
2
Genomic DNA Extraction and Sequencing
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Total genomic DNA of 1 µg was obtained from each of the three species and fragmented using an S220 ultra sonicator (Covaris, Woburn, USA). The library preparation was performed using a MGIEasy DNA library prep kit (MGI, Shenzhen, China) according to the manufacturer’s instructions. The library was quantified with a QauntiFluor ssDNA System (Promega, Madison, USA). Sequencing was performed for a MGI paired-end library, and 150 bp paired-end reads were generated using a MGISEQ-2000 platform (MGI, Shenzhen, China).
3
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.
4
Whole Transcriptome Sequencing Library Prep
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Total RNA (500 ng) was used to prepare whole transcriptome sequencing libraries. Whole transcriptome RNA was enriched by depleting ribosomal RNA (rRNA), and a complete transcriptome sequencing library was generated using the MGIEasy RNA Directional Library Prep Kit (MGI) according to the manufacturer’s instructions. After rRNA depletion, the remaining RNA was fragmented into small pieces by treatment with divalent cations under elevated temperatures. Then, the cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers. Strand specificity was achieved in RT directional buffer, followed by second-strand cDNA synthesis. An additional A base was added to the cDNA fragments and then an adapter was ligated. The products were then purified and enriched by PCR to create the final cDNA library.
The double-stranded library was quantified using the QauntiFluor ONE dsDNA System (Promega, Madison, WI, USA) and 330 ng in a total volume of 60 μl or less. The library was cyclized at 37 °C for 60 min, digested at 37 °C for 30 min, and then the circularization product was cleaned up. The library was incubated at 30 °C for 25 min with DNA nanoball (DNB) enzyme. Finally, the library was quantified using the QauntiFluor ssDNA System (Promega) and sequenced using the MGIseq system (MGI) to generate 150 bp paired-end reads.
The double-stranded library was quantified using the QauntiFluor ONE dsDNA System (Promega, Madison, WI, USA) and 330 ng in a total volume of 60 μl or less. The library was cyclized at 37 °C for 60 min, digested at 37 °C for 30 min, and then the circularization product was cleaned up. The library was incubated at 30 °C for 25 min with DNA nanoball (DNB) enzyme. Finally, the library was quantified using the QauntiFluor ssDNA System (Promega) and sequenced using the MGIseq system (MGI) to generate 150 bp paired-end reads.
5
RNA-seq Library Preparation Protocol
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RNA was extracted using the RiboEx reagent (GeneAll, Seoul, South Korea). RNA was prepared for the mRNA sequencing library using the MGIEasy RNA Directional Library Prep Kit (MGI) according to the manufacturer’s instructions. The products are then purified and enhanced by PCR to form the final cDNA library. The double-stranded library is measured using the QauntiFluor ONE dsDNA System (Promega). The library is followed by the cleanup of circularization products. The QauntiFluor ssDNA System (Promega) was used to quantify the library. The constructed library was then sequenced on the MGIseq system (MGI). All datasets created and analyzed in this study are deposited in the NCBI’s Gene Expression Omnibus (GSE230271).
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