Open Science / Protocols

Protocols

How the MuLTEE is run and measured, from daily settling selection to phenotyping: 15 protocols, written and maintained by the lab and updated as methods change.

Analysis pipelines

• Breaking up and crushing clusters: collected methods

  By Various authorsVersion 1Updated 2026-10-07

  The different ways clusters are broken up or crushed, collected in one place from the transformation, spheroplast, cell volume and ploidy protocols.

• Daily growth and settling selection

  By Various authorsVersion 1Updated 2026-10-07

  Standard 24 h growth (used for PAs and PMs - POs use YPG where glycerol provided as a direct substitution for dextrose) in YPD followed by daily settling selection, used to propagate snowflake yeast populations.

• Archiving cultures at −80 °C

  By Various authorsVersion 1Updated 2026-10-07

  Making 35% glycerol freezer stocks of populations or strains.

• Reviving strains from frozen stock

  By Various authorsVersion 1Updated 2026-10-07

  Bringing strains back from −80 °C stocks and re-acclimating them to full cluster size with settling selection.

• Genomic DNA extraction (MasterPure Yeast kit)

  By Various authorsVersion 1Updated 2026-10-07

  Purifying genomic DNA from a liquid culture pellet or a single colony.

• Isolating individual spheroplasts for single-cell RNA extraction

  By Various authorsVersion 1Updated 2026-10-07

  Getting a clean suspension of single spheroplasts from clusters for single-cell RNA work, with every step at 0–4 °C.

• RNA extraction (TRI Reagent and Direct-zol)

  By Various authorsVersion 1Updated 2026-10-07

  Bulk RNA extraction using bead-beating in TRI Reagent and Direct-zol columns.

• Colony selection from plates

  By Various authorsVersion 1Updated 2026-10-07

  Recognising true snowflake clones (“volcano” or “donut” colonies) when picking from plates.

• Measuring cell aspect ratio

  By Various authorsVersion 1Updated 2026-10-07

  Calcofluor White staining and ×40 imaging for Cellpose-based cell shape measurements.

• Measuring cell volume

  By Various authorsVersion 1Updated 2026-10-07

  Crushing clusters to a single layer, then Calcofluor staining and imaging to estimate cell volume as an ellipsoid.

• Measuring cluster size

  By Various authorsVersion 1Updated 2026-10-07

  Imaging cluster cross-sectional area after 5 days of settling selection, for size segmentation.

• Measuring ploidy (propidium iodide staining)

  By Various authorsVersion 1Updated 2026-10-07

  Staining DNA with propidium iodide and imaging to measure ploidy against known diploid and tetraploid controls.

• CRISPR-Cas9 knock-in at the ChrXIII Int8 locus

  By Various authorsVersion 1Updated 2026-10-07

  Single-locus knock-ins (e.g. GFP labelling) in the Y55 background, with Golden Gate–built Cas9 and donor plasmids (constitutive TEF or inducible promoter).

• Lithium acetate/PEG transformation of snowflake yeast

  By Various authorsVersion 1Updated 2026-10-07

  The standard LiOAc/PEG transformation adapted for multicellular clusters, for plasmids, PCR products or CRISPR constructs.

• Modified Lithium acetate/PEG transformation of snowflake yeast

  By Dung LacVersion 1Updated 2026-10-20

  Modified LiOAc/PEG transformation adapted for multicellular clusters, for plasmids, PCR products for better efficiency

Last updated 2026-10-20. Questions about a protocol: see who to write to.