This BBC_2024__README.txt file was generated on 2024-09-25 by Beatriz Baselga Cervera GENERAL INFORMATION 1. Title of Dataset and code: Data from: Phenotypic stutter steps in experimentally evolved multicellularity. 2. Author Information Corresponding Investigator Name: Dr Beatriz Baselga-Cervera Institution: University of Minnesota Twin Cities, Minnesota, US. Email: bbaselga@umn.edu; beabaselga@gmail.com Co-investigator 1 Name: Dr Nahui Olin Medina-Chávez Institution: University of Minnesota Twin Cities, Minnesota, US. Email: nmedinac@umn.edu Co-investigator 2 Name: Dr Michael Travisano Institution: University of Minnesota Twin Cities, Minnesota, US. Email:travisan@umn.edu 4.Data collectors: Dr Beatriz Baselga-Cervera & Dr. Nahui Olin Medina-Chávez. 5. Date of data collection: 2023-2024 6. Geographic location of data collection: Saint Paul, US 5. Funding sources that supported data collection: Fundación Alfonso Martín Escudero, Madrid, Spain (BBC). 6. Recommended citation for this dataset: Baselga-Cervera et al. (2024), Data from: Phenotypic stutter steps in experimentally evolved multicellularity. Zenodo. Data set. DATA & FILE OVERVIEW 1. Description of dataset In this study, we address whether stochastic phenotypic switching can shape biological diversity contributing to evolutionary change across the transition from single cells to multicellular clutters in Saccharomyces cerevisiae multicellular yeast system. Population's characterization was conducted with a Coulter Counter multisize 4, and a FlowCam 3, by colony morphology, under the optic microscope, via ACE2 gene sequencing and RNA sequencing. The populations studied were the genetically uniform diploid wild-type Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockouts, and strains containing the missense mutation (ACE2 c.1934 A>T). 2. File list: Coulter Counter size distribution data: File 1 name: File_1_Coulter_Counter_Counts_20h.csv File 1 description: Size distributions of Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockout, and strains containing the missense mutation (ACE2 c.1934 A>T) in YPD at 20hgrowth. File 2 name: File_2_Coulter_Counter_Counts_24h.csv File 2 description: Size distributions of Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockout, and strains containing the missense mutation (ACE2 c.1934 A>T) in YPD at 24h growth. File 3 name: File_3_Coulter_Counter_Counts_48h.csv File 3 description: Size distributions of Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockout, and strains containing the missense mutation (ACE2 c.1934 A>T) in YPD at 48h growth. File name: File_4_Coulter_Counter_Counts_Constructed_strains_diversity.csv File 4 description: Size distributions of the constructed ACE2 knockout and a strain containing the homozygous missense mutation (ACE2 c.1934 A>T) in YPD at 24h growth. Size distributions were obtained from populations before (initial) and after gravitational selection of five resuspended colonies from three isolates per strain. File 5 name: File_5_Coulter_Counter_Counts_Selection_Experiment.xlsx File 5 description: Size distributions of C1W8.1 and C1W8.2 multicellular evolved strains in YPD at 24h growth. Size distributions from the selection experiment for small-size particles by plating the top fraction of the population after gravitational selection and maintaining lineages based on colony morphology. FlowCam data: File 6 name: File_6_Rawdata_Flowcam_all.csv File 6 desciption: FlowCam data from Saccharomyces cerevisiae Y55 strain clones, C1W8.1 and C1W8.2 multicellular evolved strains, constructed ACE2 gene knockouts, and strains containing the missense mutation (ACE2 c.1934 A>T) in YPD at 24h growth. Data generated statistically: File 7 name: File_7_C1W8.2_overlapPairs_Selection_Experiment.csv File 7 description: overlapping indexes (η) of the KDE distributions were computed using the R-package ‘overlapping’ from the Coulter Counter data of the C1W8.2 derived strain over the selection experiment. File 8 name: File_8_C1W8.1_overlapPairs_Selection_Experiment.csv File 8 description: overlapping indexes (η) of the KDE distributions were computed using the R-package ‘overlapping’ from the Coulter Counter data of the C1W8.1 derived strain over the selection experiment. File 9 name: File_9_ overlapPairs_Constructed_strains_diversity.xlsx File 9 description: overlapping indexes (η) of the KDE distributions were computed using the R-package ‘overlapping’ from the Coulter Counter data of the constructed ACE2 knockout and a strain containing the homozygous missense mutation (ACE2 c.1934 A>T) in YPD at 24h growth. Size distributions were obtained from populations before (initial) and after gravitational selection of five resuspended colonies from three isolates per strain. Pictures: File 10 name: File_8_Colonies_Pictures_Constructed_strains.zip File 10 description: constructed ACE2 knockout and a strain containing the homozygous missense mutation (ACE2 c.1934 A>T) YPD agar plates and magnifications when dilution plating the small size fraction of the population. ARN data File 11 name: File_11_rnaseq-final-results-Top_v_Bottom.xlsx File 11 description: RNA analyses top vs. bottom phenotypic subdistributions' final results. The Top subdistribution is used as control. METHODOLOGICAL INFORMATION Strains: ancestral wildtype (Y55 strains), C1W8.1 and C1W8.2 multicellular derived strains isolated after 60 days of selection in YPD media, constructed ACE2 gene knockouts, and strains containing the ACE2 missense mutation (ACE2 c.1934 A>T). Media: Growth media used in this study were Yeast Peptone Dextrose media (YPD; 1% (v/w) yeast extract, 2% (v/w) peptone, 2% (v/w) D-glucose, pH 5.8) and Standard minimal (SD; Yeast nitrogen base with amino acids (YNB w/AA) 6.7 g L-1, 0.5% (v/w) D-glucose). Phenotypic characterization of the different strains was conducted in a Coulter Counter Multisizer 4 and FlowCam® 3.0 Fluid Imaging Technologies, colony morphology, and optic microscopy. Replicate populations of different individual isolates per strain were analyzed to obtain the population distributions in YPD media. RNA was extracted using an Invitrogen® PureLink RNA Mini Kit. Three of four extracted samples per treatment with the highest RNA integrity score were submitted for TrueSeq Stranded RNA-Seq. 3. Detailed description Coulter Counter size distribution data of all the populations: File 1 name: File_1_Coulter_Counter_Counts_20h.csv File 1 description: strains naming convention; strain_Isolate_run.pseudoreplicate. Strains: ace2x2m=strains containing the ACE2 missense mutation (ACE2 c.1934 A>T); ace2x2= ACE2 knockout; C1W8.1= C1W8.1 evolved multicellular strain; C1W8.2= C1W8.2 evolved multicellular strain; Y55= ancestral strain. § Page 1: Column 1: Volumen (um3) Column 2: Diameter (um2) Columns 3 to the last column: strains counts. File 2 name: File_2_Coulter_Counter_Counts_24h.csv File 2 description: strains naming convention; strain_Isolate_run.pseudoreplicate. Strains: ace2x2m=strains containing the ACE2 missense mutation (ACE2 c.1934 A>T); ace2x2= ACE2 knockout; C1W8.1= C1W8.1 evolved multicellular strain; C1W8.2= C1W8.2 evolved multicellular strain; Y55= ancestral strain. § Page 1: Column 1: Volumen (um3) Column 2: Diameter (um2) Columns 3 to the last column: strains counts. File 3 name: File_3_Coulter_Counter_Counts_48h.csv File 3 description: strains naming convention; strain_Isolate_run.pseudoreplicate. Strains: ace2x2m=strains containing the ACE2 missense mutation (ACE2 c.1934 A>T); ace2x2= ACE2 knockout; C1W8.1= C1W8.1 evolved multicellular strain; C1W8.2= C1W8.2 evolved multicellular strain; Y55= ancestral strain. § Page 1: Column 1: Volumen (um3) Column 2: Diameter (um2) Column 3 to the last column: strains counts. File_4_Coulter_Counter_Counts_Constructed_strains_diversity.csv File 4 description: strains naming convention; strain_Isolate_colony_run.pseudoreplicate. Strains: ace2x2m=strains containing the ACE2 missense mutation (ACE2 c.1934 A>T); ace2x2= ACE2 knockout. § Page 1: Column 1: Volumen (um3) Column 2: Diameter (um2) Column 3 to the last column: strains counts. File_5_Coulter_Counter_Counts_Selection_Experiment.xlsx File 5 description: strains naming convention; strain_colony.phenotype_selection.cycle_run.pseudoreplicate. Strains: C1W8.2= C1W8.2 evolved multicellular strain and C1W8.1= C1W8.1 evolved multicellular strain. § Page 1: Column 1: Volumen (um3) Column 2: Diameter (um2) Column 3 to the last column: strains counts. File 6 name: File_3_Rawdata_Flowcam_all.csv File 6 description: strains naming convention; ace2_isolate= ACE2 knockout; Ace2m_isolate= strain containing the ACE2 missense mutation (ACE2 c.1934 A>T); c1w82_isolate=C1W8.2 evolved multicellular strain; c1w81_isoalte= C1W8.1 evolved multicellular strain; Y55_isolate=ancestral strain. § Page 1: Column 1: Particle ID Column 2: Area ABD Column 3: Aspect Ratio (Width/Length) Column 4: Circle Fit Column 5: Area base Diameter (ABD) Column 6: Equivalent Spherical Diameter (ESD) Column 7: Elongation Column 8: Perimeter Column 9: Roughness Column 10: Volume ABD-based Column 11: Volume ESD-based Column 12: Width Column 13: Source. Name of the sample. File 7 name: File_7_C1W8.2_overlapPairs_Selection_Experiment.csv File 7 description: C1W8.2 _lineage_selection.cycle= C1W8.2 evolved multicellular strain, lineage (A=ancestral, M1= rugose colony morphology lineage 1, M2= rugose colony morphology lineage 2 , M3= rugose colony morphology lineage 3 , U1= rough colony morphology lineage 1, U2= rough colony morphology lineage 2, U3= rough colony morphology lineage 3) and selection cycle (0, 1, 2 and 3). § Page 1: Column 1: Var1= strain 1 Column 2: Var2= strain 2 Column 3: overlap value of both strains compared. File 8 name: File_8_C1W8.1_overlapPairs_Selection_Experiment.csv File 8 description: C1W8.1 _lineage_selection.cycle =C1W8.1 evolved multicellular strain, lineage (A=ancestral, M1= rugose colony morphology lineage 1, M2= rugose colony morphology lineage 2 , M3= rugose colony morphology lineage 3 , U1= rough colony morphology lineage 1, U2= rough colony morphology lineage 2, U3= rough colony morphology lineage 3) and selection cycle (0, 1, 2 and 3). § Page 1: Column 1: Var1= strain 1 Column 2: Var2= strain 2 Column 3: overlap value of both strains compared. File 9 name: File_9_overlapPairs_Constructed_strains_diversity.xlsx File 9 description: variables naming convention; strain _isolate_colony.number. Strains; ace2x2m=strains containing the ACE2 missense mutation (ACE2 c.1934 A>T); ace2x2= ACE2 knockout. Isolate; 1,2 and 3. Colony.number; Initial=initial population and colony number (1,2,3,4 and 5). § Page 1: Column 1: Var1= strain 1 Column 2: Var2= strain 2 Column 3: overlap value of both strains compared. File 10 name: File_10_Colonies_Pictures_Constructed_strains.zip File 10 description: colony pictures by strain. Strains: Strains; ace2x2 m=strains containing the ACE2 missense mutation (ACE2 c.1934 A>T); ace2x2= ACE2 knockout. File 11 name: File_11_rnaseq-final-results-Top_v_Bottom.xlsx File 11 description: § Page 1: Column 1: number Column 2: ID Column 3: protID Column 4: gene_symbol Column 5: chr Column 6: chr_latin Column 7: location Column 8: baseMean Column 9: log2FoldChange Column 10: lfcSE Column 11: stat Column 12: pvalue padj Column 13: test Column 14: log10padj Column 15: log10baseMean Column 16: blast_pident Column 17: transcript_length Column 18: blast_evalue Column 19: blast_bitscore Column 20: rnaID Column 21: feature Column 22: accession Column 23: strain Column 24: gene_accession