RNA was extracted using the RNeasy Mini Kit (QIAGEN), including the optional DNase step to prevent genomic DNA contamination. RNA integrity was assessed using RNA 6000 Nano Chips on a 2100 Bioanalyser (Agilent). Only samples with non-degraded RNA (RIN ≥ 7) were used for sequencing. Sequencing of RNA samples was performed by deCODE Genetics, Iceland. Preparation of indexed cDNA sequencing libraries was carried out using the TruSeq poly-A mRNA method (Illumina). Briefly, poly-A mRNA transcripts were captured from total RNA using poly-T beads, before cDNA was generated using random hexamer priming. Paired-end sequencing (2 × 100 cycles) of indexed cDNA libraries was then carried out on a HiSeq 2500 machine (Illumina), generating at least 50 million reads (101 base pairs) per sample. Sequencing was performed using v4 SBS and Cluster Kits (Illumina). One sample failed the library generation step and was excluded from the study. After sequencing the indexed samples were demultiplexed before generation of FASTQ files for analysis. Raw data files are available from the European Nucleotide Archive (http://www.ebi.ac.uk/ena ) under the study accession number PRJEB12995.
Truseq poly a mrna method
Manufactured by Illumina
The TruSeq poly-A mRNA method is a laboratory technique used for the isolation and purification of messenger RNA (mRNA) from total RNA samples. This method selectively enriches for polyadenylated mRNA molecules, which represent the actively translated portion of the transcriptome. The core function of this method is to provide a standardized approach for extracting mRNA from complex RNA samples, enabling downstream applications such as gene expression analysis and RNA sequencing.
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2 protocols using truseq poly a mrna method
1
RNA-seq Library Preparation and Sequencing
2
Striatum RNA-seq of Injected Mice
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Total RNA (500 ng) from the striatum of injected mice was extracted using the RNeasy Mini Kit (QIAGEN; 74104) and underwent DNase treatment to prevent genomic DNA contamination. RNA integrity was assessed using RNA 6000 Nano Chips on a 2100 Bioanalyzer (Agilent; 5067-1511). Samples with non-degraded RNA (RIN ≥ 8.5) were used for sequencing. Preparation of indexed cDNA sequencing libraries was carried out using the TruSeq poly-A mRNA method (Illumina; 20020594). Briefly, poly-A mRNA transcripts were captured from total RNA using poly-T beads and cDNA was generated using random hexamer priming. Paired-end sequencing (2 × 50 cycles) of indexed cDNA libraries was then carried out on a HiSeq 2500 machine (Illumina), generating at least 50 million reads per sample. Sequencing was performed using v4 SBS and Cluster Kits (Illumina; FC-401-4002). RNA sequencing data are available at Gene Expression Omnibus with accession number GSE165667.
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