Materials

• Saccharomyces cerevisiae ATCC™ 9763™ Culti-Loops™
• Refrigerated centrifuge (with capacity for 50 ml Flacon tubes)
• Mini-centrifuge
• Incubator or water bath (capacity for 30°C)
• Ice and ice bucket
• Vortex
• 1.5 and 2 ml Eppendorf tubes
• 50 ml Falcon tubes
• 1x phosphate buffered saline (PBS)
• TRIzol
• Chloroform
• Isopropanol
• Ethanol
• Zymolyase resuspended to 1000 U/ml
• TE buffer (1 mM EDTA, pH 8.0)
• Y1 buffer - this can be purchased as part of the QIAGEN genomic buffer set or prepared according to the manufacturer’s manual (page 62).

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. Collect 15 ml of yeast culture in a 50 ml Falcon tube and centrifuge for 10 minutes at 1500 x g at 4°C.

3. Discard the supernatant and add 15 ml of PBS 1x to the cell pellet and invert the tube to resuspend.

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

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 2 ml of Y1 buffer and 100 µl of zymolyase resuspended to 1000 U/ml, to the yeast pellet and mix thoroughly by pipetting. Note: Add 2 ml of Y1 buffer and 100 µl of zymolyase resuspended to 1000 U/ml, to the yeast pellet and mix thoroughly by pipetting.

8. Incubate at 30°C for 30 minutes.

9. Centrifuge for 5 minutes at 300 x g at 4°C.

10. Discard the supernatant and retain the pellet.

11. Add 1 ml of TRIzol to the pellet and mix with a pipette.

12. Transfer the solution to a 2 ml Eppendorf tube.

13. Incubate at room temperature for 5 minutes.

14. Add 200 µl of chloroform and vortex for 10 seconds.

15. Incubate at room temperature for 5 minutes.

16. Centrifuge for 5 minutes at 2000 x g at 4°C.

17. Discard the pellet and transfer the supernatant into a new 2 ml Eppendorf tube and add 400 µl of chloroform.

18. Vortex for 10 seconds and incubate 5 minutes at room temperature.

19. Centrifuge for 5 minutes at 2000 x g at 4°C.

20. Discard the pellet and transfer the supernatant to a new 1.5 ml Eppendorf tube.

21. Add 0.5 volume of isopropanol.

22. Incubate on ice for 15 minutes.

23. Centrifuge for 5 minutes at 2000 x g at 4°C.

24. Discard the supernatant.

25. Wash the pellet with 1 ml of ice-cold 70% ethanol.

26. Centrifuge for 2 minutes at 2000 x g at 4°C.

27. Repeat steps 23–25.

28. Discard the supernatant and allow the pellet to air-dry for a couple of minutes. Note: Do not over-dry the pellet as it might be hard to resuspend.

29. Elute the pellet in 100 µl of TE and mix by pipetting.

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

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 panel. 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.

Results

• Yield: 200—300 µg
• OD 260/280: 2.22
• OD 260/230: 2.02

• Agilent Bioanalyzer RNA 6000 Nano Kit RIN: 9.7

Change log

Version	Change
v2, 18th August 2020	Added zymolyase concentration
v1, 28th March 2019	Initial protocol publication