Transcriptomic analysis was performed as described previously [21 (link)]. Total RNA from MCF-7 and MCF-7SCs was isolated using a TRIzol kit (Invitrogen, Carlsbad, CA, USA) and subjected to RNA sequencing (RNA-seq) by Macrogen (Seoul, Korea). Signaling pathways were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatic Annotation Server (KAAS).
Rna seq
Manufactured by Macrogen
RNA-seq is a powerful sequencing technology that enables the analysis of the complete transcriptome. It provides a comprehensive view of the RNA molecules present in a biological sample, allowing researchers to study gene expression, identify novel transcripts, and detect alternative splicing events.
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5 protocols using rna seq
1
Transcriptomic Analysis of MCF-7 Cells
2
RNA-Seq Analysis of H. mediterranei under Stress Conditions
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Culture conditions for RNA-Seq were: Hm-DM in the presence of 20 mM ammonium (control condition), Hm-DM with 8 mM H2O2, Hm-CS at 48 h, and 0.5 M lithium (LiCl). Three independent replicates of H. mediterranei R4 cells from each condition were grown, harvested, and RNA was isolated as detailed above. Following this, the transcriptome of 12 samples was analyzed by RNA-Seq (Macrogen Inc. Seoul, Korea). The RNA-Seq library preparation and sequencing were performed using the TruSeq Stranded Total RNA with NeBNext rRNA Depletion Kit (New England BioLabs, Ipswich, Massachusetts, USA) to construct the cDNA libraries and subsequently sequenced using an Illumina NovaSeq 6000 system (Illumina, Cambridge, United Kingdom) according to manufacturer's guidelines. The libraries were checked and quantified using an Agilent Technologies 2100 Bioanalyzer by qPCR following qPRC Quantification Protocol Guide (Agilent, Santa Clara, USA).
3
Illumina RNA-Seq transcriptome profiling
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Global messenger RNA sequencing was performed by Macrogen’s “RNA-seq” service in South Korea. In summary, Illumina TruSeq RNA libraries were prepared, and 100 bp (Paired-End) sequences were obtained from each of them on a HiSeq 4000 platform. Between 12 and 14 million reads per library were obtained. The data are deposited at the NCBI, BioProject ID: PRJNA739193.
4
Transcriptome Analysis of MCF-7 and MCF-7/SC Cells
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Transcriptome analysis of MCF−7 and MCF−7/SC cells was performed in a recent study conducted in our laboratory [22 (link)]. Total RNA was extracted from MCF−7 and MCF−7/SC cells using a TRIzol kit (Invitrogen, Carlsbad, CA, USA). An Illumina TruSeq mRNA Sample Prep kit (Illumina, San Diego, CA, USA) was used to create the RNA library. To prepare for sequencing, mRNA was purified using magnetic beads with Poly−T oligo attachments. RNA−Seq was performed by Macrogen, Inc. (Seoul, Korea), in accordance with the manufacturer’s instructions. A genomic DNA reference (UCSC hg19) was used to apply the cDNA fragment obtained through the RNA sequencing. The HISAT2 program was used to align the read mapping through the Bowtie 2 aligner. Signaling pathways were investigated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Automatic Annotation Server (KAAS).
5
RNA-seq Analysis of hSOD1 Mutant Cells
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Three sets of NSC34 cells (transfected with wild-type or G93A mutant hSOD1) were grown and harvested with each set from a separate passage of single cell line. Following subcellular fractionation, transcriptomes of 12 samples were analysed by RNA-seq (Macrogen Inc. Seoul, Korea), as described previously. Briefly, 1 μg of the total RNA was analysed using the TruSeq RNA library kit to construct the cDNA libraries. The protocol included polyA-selected RNA extraction, RNA fragmentation, random hexamer primed reverse transcription and 100 nt paired-end sequencing using an Illumina HiSeq2000 (Illumina, San Diego, CA, USA). The libraries were quantified using quantitative real-time polymerase chain reaction (qPCR) according to the qPCR Quantification Protocol Guide. An Agilent Technologies 2100 Bioanalyzer was used for the qualification.
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