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  • rRNA contamination and streptavidin beads

    Hello everyone!

    I recently prepared a RNA library (RNA extracted from cultured fibroblasts) and ran it on a NextSeq 500, using the Illumina TruSeq Stranded Total RNA Kit and using the Illumina Ribo-Zero rRNA Depletion Kit (legacy version, i.e. streptavidin pull-down) for rRNA depletion.

    In the results, I noticed that ~90% of all sequencing reads aligned to the ribosomal contig and therefore I suspected rRNA contamination. I then found out that the rRNA removal beads (Illumina) had been stored at -20°C for roughly 2 years. According to Illumina, storing these beads not at -20°C (instead of the recommended +4°C) could be a reason for rRNA contamination.

    My question is: could the freezing impair the binding capacity of the beads or has anybody made similar experiences?

    Thank you very much in advance for any help and best regards!

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