Material stored in RNAlater (ThermoFisher Scientific) was thawed on ice, diluted with 0.7 mL nuclease-free PBS and pelleted by centrifugation at 6 k × g for 5 min at 4°C. RNA was extracted from the resulting pellet using TRIzol (ThermoFisher Scientific) according to the manufacturer’s instructions. RNase-free glycogen (10 μg) was used as a carrier during the precipitation phase to improve RNA yield. RNA yield was measured using the Qubit RNA high sensitivity kit (ThermoFisher Scientific), and sample purity and integrity was verified by TapeStation (Agilent) electrophoresis using RNA ScreenTape. To process samples for metantranscriptomic sequencing, total RNA was depleted of rRNA using the QIAseq FastSelect 5S/16S/23S kit (beta version, Qiagen), sequencing libraries were generated using the SMART-Seq Stranded for total RNA-seq kit (Takara) and 75 base pair, paired end reads were generated using an Illumina MiSeq v3 platform.
Smart seq stranded for total rna seq kit
Manufactured by Takara Bio
The SMART-Seq Stranded for total RNA-seq kit is a laboratory tool designed for high-quality total RNA sequencing. It provides a streamlined workflow for converting total RNA into a sequencing-ready library.
Automatically generated - may contain errors
Lab products found in correlation
Rnalater, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Qubit rna high sensitivity kit, by Thermo Fisher Scientific (1 mentions)
Qiaseq fastselect 5s 16s 23s kit, by Qiagen (1 mentions)
Miseq v3 platform, by Illumina (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
Qubit flex fluorometer, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 sequencer, by Illumina (1 mentions)
Direct zol rna microprep kit, by Zymo Research (1 mentions)
Tapestation system, by Agilent Technologies (1 mentions)
2 protocols using smart seq stranded for total rna seq kit
1
Metagenomic RNA Extraction and Sequencing
2
RNA Extraction and RNA-seq Library Prep
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RNA was extracted from 15 µl of cell lysate using the Direct-zol RNA Microprep Kit (Zymo Research) following the manufacturer's instructions and including DNase treatment for 15 min at room temperature. Samples were eluted with 15 µl nuclease-free water. The RNA integrity numbers (RIN) of the samples were between 9.9 and 10.0, measured using High Sensitivity RNA ScreenTape (TapeStation system, Agilent). RNA was quantified using a Qubit Flex fluorometer (Thermo Fisher Scientific). Libraries were prepared using the SMART-seq Stranded for total RNA-seq kit (Takara) from 5 ng of RNA and sequenced on the Illumina NextSeq 500 sequencer in the Genomics Facility Basel (Department of Biosystems Science and Engineering (D-BSSE), ETH Zürich).
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