Total RNA from gastrocnemius muscle was isolated using the RNeasy Mini Kit (74,106, Qiagen). Quantification of RNA was determined using a spectrophotometer (Nanodrop 8000, Thermo Scientific), and integrity was measured using a Bioanalyzer 2100 (Agilent). Total RNA sequencing libraries were prepared by the Single-Cell Omics platform at the Center for Basic Metabolic Research using the Illumina TruSeq Stranded total RNA Gold protocol (Illumina). The total RNA (380 ng) was depleted of rRNA by RiboZero beads, fragmented, and cDNA was synthesized using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). cDNA was adenylated to prime for adapter ligation, and after a clean-up using AMPure beads (Beckman coulter), DNA fragments were amplified using PCR, followed by a final clean-up. Libraries were quality controlled using a Bioanalyzer instrument (Agilent Technologies) and subjected to 38-bp paired-end sequencing on a NextSeq 500 system (Illumina).
Ribozero beads
Manufactured by Thermo Fisher Scientific
RiboZero beads are a laboratory reagent used for the depletion of ribosomal RNA (rRNA) from total RNA samples. They provide a simple and efficient method for removing abundant rRNA, which allows for the enrichment of other RNA species, such as mRNA, for downstream applications like RNA sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Truseq stranded total rna gold protocol, by Illumina (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Ampure bead, by Beckman Coulter (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Nanodrop 8000, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
2 protocols using ribozero beads
1
Gastrocnemius Muscle RNA Sequencing
2
Total RNA-Seq Library Preparation
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Total RNA was isolated using Trizol (Thermo Fisher Scientific) followed by purification with an RNeasy mini kit (Qiagen) according to the manufacturer's protocol. Total RNA-Seq libraries were prepared using the Illumina TruSeq Stranded total RNA Gold protocol (Illumina). The total RNA (500 ng) was depleted of rRNA by RiboZero beads, fragmented, and cDNA was synthesized using SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). cDNA was adenylated to prime for adapter ligation, and after a cleanup using AMPure beads (Beckman Coulter), DNA fragments were amplified using PCR followed by a final cleanup. Libraries were quality controlled using a Bioanalyzer instrument (Agilent Technologies).
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