5 μl of TURBO DNase treated RNA was polyadenylated with E. coli PAP (E-PAP, ThermoFisher Scientific) in 20 μl reactions, containing 1x reaction buffer, 2.5 mM MnCl2; 0.4 U E-PAP and 0.8 U RiboLock RNase inhibitor (ThermoFisher Scientific) for 30 min at 30°C. Reactions were then purified using a PureLink micro RNA purification kit (Ambion) and eluted using 22 μl RNase-free water. PureLink eluates were rRNA depleted using a down-scaled reaction of the Ribo-Zero Gold rRNA Removal Kit for Yeast (Illumina). That is, 18 μl of RNA was incubated with 2 μl of reaction buffer, 2 μl of removal solution and 8 μl of water for 10 min at 65°C, and put to RT for 5 min. This reaction was cleared using 65 μl of rRNA magnetic beads for 5 min at RT and 5 min at 50°C. Final samples were precipitated using NaOAc and ethanol with 20 μg of glycogen as carrier and resuspended in 10 μl of RNA storage solution.
Purelink micro rna purification kit
Manufactured by Thermo Fisher Scientific
The PureLink micro RNA Purification Kit is a lab equipment product designed for the isolation and purification of microRNA (miRNA) from various sample types, including cells, tissues, and body fluids. The kit utilizes a silica-based membrane technology to selectively capture and purify miRNA molecules, enabling their downstream analysis and applications.
Automatically generated - may contain errors
2 protocols using purelink micro rna purification kit
1
Polyadenylation and rRNA Depletion of RNA
2
Purification and Sequencing of 4tU-labeled RNA
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RNA quality of total and purified 4tU labeled RNA was tested and quantified using a Bioanalyzer (Agilent). Depending on the sample 200-350 ng of RNA was used to prepare libraries. To generate pA- data, an aliquot of the 2 min 4tU labeled RNAs was in vitro polyadenylated using Escherichia coli poly(A) polymerase (AM1350; ThermoFisher) according to the manufacturer’s instructions. Reactions were then purified using PureLink micro RNA purification kit from Ambion (12183018A) and ribo-depleted using Ribo-Zero Gold rRNA Removal kit for yeast (MRZY1306) from Ilumina according to the manufacturer’s instructions. All libraries were then prepared using Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit REV (016.96, Lexogen GmbH) for Illumina and sequenced at Vienna Biocenter Core Facilities (Vienna, Austria) multiplexing 32 samples on a HiSeqV4 SR50 (single end 50nt reads) run and using the QuantSeq REV specific primer CSP. An overview of samples, read quality control and mapping statistics are provided in Table S1 and Table S2 .
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