Materials

• Saccharomyces cerevisiae ATCC™ 9763™ Culti-Loops™
• Zirconium beads
• Ambion RNAqueous™ Total RNA Isolation Kit
• Centrifuge (with capacity for 1.5 ml and 5 ml tubes)
• Vortex
• 1.5 ml Eppendorf tubes
• 5 ml Eppendorf tubes
• 1x phosphate buffered saline (PBS)
• TE buffer (1 mM EDTA, pH 8.0)

Method

1. Grow 1 Culti-Loop™ of yeast in 150 ml of YPD media, overnight at 30°C with agitation (150 rpm). Measure the OD600 and dilute in YPD media to an OD600 of 0.2. Continue the growth until an OD600 of 0.6 is reached at around 2-3 hours.

2. Add 3 ml of yeast culture to a 50 ml Falcon tube and centrifuge for 10 minutes at 1500 x g.

3. Discard the supernatant and add 3 ml of PBS 1x and invert the tube to resuspend the pellet.

4. Centrifuge the tube for 10 minutes at 1500 x g.

5. Discard the supernatant and retain the pellet.

6. Optional Step: At this stage the protocol can be paused, and the pellet stored at -80°C.

7. Add 300 µl of lysis/binding solution and transfer the lysate to a 1.5 ml Eppendorf tube.

8. Take one tube of BeadBugTM zirconium beads and pour the beads into the lysate solution.

9. Vortex at maximum speed for 1 minute and then incubate without agitation for 1 minute at room temperature.

10. Repeat Step 8, three more times.

11. Continue the extraction following the RNAqueous™ Kit protocol (pages 13–15, steps 1–6). For the elution, we recommend a first elution with 50 µl and a second elution with 30 µl.

12. Aliquot the sample as necessary and store aliquots at -80°C.

Results

Yield: 20–50 µg OD 260/280: 2.17 OD 260/230: 1.87

Agilent Bioanalyzer RNA 6000 Nano Kit, RIN: 8.0

Sequencing performance

Libraries for nanopore sequencing were prepared from 50 ng of total RNA ±200 pg of ERCC RNA Spike-In Mix, using the PCR-cDNA Sequencing Kit.

• Read length profile:

Figure 1. The length distribution of reads that align to S. cerevisiae and the ERCC Spike-In Mix. Panel A: the length distribution of reads that align to the S. cerevisiae reference genome. Panel B: the proportion of the ERCC transcript covered by a read aligning to that transcript.. The observed lengths of the reads that aligned to the ERCC panel show the majority of reads cover almost the entire transcript (as is expected, as the PCR-cDNA Sequencing Kit enriches for full-length RNA template molecules). This suggests that length distribution of reads in Panel A is representative of the length of the RNA molecules present in the sample and is not being biased during the library preparation process. The Spearman’s rank correlation coefficient (rho) and the coefficient of determination (r^2) for the ERCC alignments were >0.95, further suggesting that there is very little bias in the library preparation and sequencing.

Change log

Version	Change
v1, 28th March 2019	Initial protocol publication