Sample preparation

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Table of contents

1. Cell growth and RNA extraction
2. Poly(A) tailing, rRNA depletion and additional RNA treatment
3. Library preparation

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Cell growth and RNA extraction

• Grow Escherichia coli K-12 MG1655 cells in rich medium (10 g tryptone, 5 g yeast extract, 5 g NaCl per liter, pH 7.2) to an OD_600nm of 0.5-0.6 (or your organism of choice in the conditions you´re interested in)
• Stabilize RNAs by adding two volumes of RNAlater (Thermo Fisher Scientific)
• Store cultures at -20°C or harvest cells by centrifugation at 4°C
• Extract RNA with the method of your choice. We (and others) had good experience with the RNeasy Kit from Qiagen

  Note: Silica-membrane columns have a cut-off size of about 200 nucleotides

• Check integrity of total RNA (Bioanalyzer)

  Note: RNA degradation has a massive influence on the results of your experiment

Poly(A) tailing, rRNA depletion and additional RNA treatment

• Perform poly(A)–tailing using the E. coli poly(A) polymerase (New England Biolabs):
  • Incubate RNAs at 70°C for 2 min and snap cool it on a pre-chilled freezer block
  • Incubate 5 µg RNA, 20 units poly(A) polymerase, 5 µl reaction buffer, 1 mM ATP for 15 min at 37°C in a total reaction volume of 50 µl.
  • Stop and clean up the reaction following the RNeasy Micro clean-up protocol (Qiagen)
  • Evaluate efficiency of poly(A)-tailing (Bioanalyzer, peaks of rRNAs)

Note: Efficiency of poly(A)-tailing is a critical step, but may not work equally well in other organisms.

• Perform rRNA depletion, e.g. using the Pan-Prokaryote riboPOOL by siTOOLs (Clean-up can also be performed using the RNeasy protocol)

Note: After depletion, only ~5% of the initial RNA input quantity are left.

• Perform additional treatment of your choice and clean-up, e.g. using the Terminator 5´-Phosphate-Dependent Exonuclease (TEX, Lucigen).

Important: Before library preparation:

1. Check extent of remaining buffer and DNA contamination that could have a negative impact on your library
2. Check RNA size and quantity (Qubit) to determine molarities as accurately as possible

Library preparation

• Libraries for Nanopore sequencing were prepared from poly(A)-tailed RNAs according to the protocols provided by ONT
• Critical steps:
  • Input quality & amount of RNA: Especially important for DRS, since you cannot get rid of excess amounts of RNA used as input. In that case, many transcripts that do not carry a motor protein are loaded onto the flowcell and overall have a negative impact on throughput/quality.
  • Reverse transcription: Maxima RT H Minus performs well and can be adjusted to high GC content
  • PCR for PCR-cDNA protocols: number of cycles adjusted to input amount (use minimal number cycles possible), extension time (adjust for longest transcripts you expect)
  • Beads to cDNA ratio (PCR-cDNA): Enrichment for longer transcripts using a 0.5 ratio
  • Molarity of ready-to-load library (check size & quantity): Avoid overloading of your flowcells! We observed that especially small fragments lead to a poor translocation spped and quality of reads.

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