Web supplement to
"Yeast Barcoders: Application of a universal donor strain collection carrying unique barcode identifiers"

Creating Barcoder strains

To create a universal barcode pool, we transformed barcodes into the HO locus of a widely used laboratory strain BY4741. The barcode cassette was constructed as described(1) with the following modifications.

• First, two 56-bp nucleotide primers, each containing (3' to 5') 18 bases of homology to the KanMX4 cassette (U1 or D1), a unique 20-bp tag sequence and an 18-bp tag priming site (U2 or D2), are used to amplify the KanMX4 module (Supplementary Fig. 1a on line). All barcode primer sequences are listed in supplemental table 1.
• In the second round of PCR, two primers (HOUP (5’-AAATCCATATCCTCATAAGCAGCAATCAATTCTATCTATACTTTAAAATGGATGTCCACGAGGTCTCT-3’) and HODN (5’-AAATTTTACTTTTATTACATACAACTTTTTAAACTAATATACACATTTTACGGTGTCGGTCTCGTAG-3’), each bearing a 50-bp of homology to the upstream or downstream region of HO gene and an 18-bp or 17-bp nucleotide complementary to the sequence of tag priming site (U2 or D2), are used to amplify the first PCR product. The PCR product of the second PCR is a 1681-bp nucleotide that contains two barcodes (uptag and downtag), the KanMX4 cassette and two 50-bp sequences homology to HO gene (supplemental figure 1a).
• These PCR products were transformed into wild type strain BY4741 using multiwell transformation protocol (http://www-sequence.stanford.edu/group/yeast_deletion_project/transprot.html). Transformed strains were selected on YPD plates with 250mg/L of G418.
• To verify correct integration of the barcode cassette, genomic DNA was prepared from the resistant strains and used as template in PCR reactions using two primers common to the KanMX4 module (KanB (5’-CTGCAGCGAGGAGCCGTAAT-3’) and KanC (5’-TGATTTTGATGACGAGCGTAAT-3’)) and two primers (A (5’-TATTAGGTGTGAAACCACGAAAAGT-3’) and D (5’-CATGTCTTCTCGTTAAGACTGCAT-3’)) flanking HO gene. Primer A is designed from region 244 base upstream of the start codon, whereas primer D is 356 base downstream of the stop codon. For correct transformation strains, the A-KanB and the D-KanC PCR reactions should produce a 566 and 1964-bp band respectively when analyzed by gel electrophoresis.
• To further confirm correct integration of barcodes, two other primers (B (5’-ACTGTCATTGGGAATGTCTTATGAT-3’) and C (5’-GAGTGGTAAAAATCGAGTATGTGCT-3’)) were designed based on the sequence within the HO gene. The A-B and C-D PCR reactions should produce no band if integration is correct.

The barcode integrated into the HO locus can be targeted to any genome locus by additional PCR followed by transformation (supplemental figure 1b). For example, to integrate barcodes into gene X locus, two primers (gene X up primer and gene X down primer), each bearing a 50-bp of homology to the upstream or downstream region of gene X and an 18-bp nucleotide complementary to the sequence of tag priming site (U2 or D2), can be used to amplify the genomic DNA of a barcode donor strain created in this study. The barcode can be then integrated into gene X locus by homologous recombination (Supplementary Fig. 1b on line).

Sequencing barcodes in the barcoded BY4741

A pool of barcoded BY4741 was constructed following standard protocol (2) . DNA of this pool was extracted and used for sequencing. The uptags and downtags were amplified using primer pair U1 (5’ GATGTCCACGAGGTCTCT 3’), U2 (5’ GTCGACCTGCAGCGTACG 3’) and D1 (5’ CGGTGTCGGTCTCGTAG 3’), D2 (5’ CGAGCTCGAATTCATCGAT 3’) respectively (Supplementary Fig. 1a). The amplicons (barcodes) were sequenced by reversible-terminator sequencing (3) using an Illumina/Solexa Genome analyzer as described by the manufacturer.

Barcoding DAmP strains by SGA

To barcode DAmP strains, each Barcoder strain was mated with a DAmP strain. The diploid double mutants were sporulated on plates at room temperature for one week. The sporulated cell mixture was grown on SGA medium (4) twice to select for MATa haploid progeny. Haploid double mutants were then selected on SGA medium containing 100 mg/L nourseothricin and 200 mg/L G418. Finally, the MATa recombinant meiotic progeny were streaked for a single colony, creating a strain collection of each allele as a DAmP mutant with a unique molecular barcode at the HO locus.

Barcoded DAmP pool construction and drug screens

Barcoded DAmP pool construction followed a standard protocol (2, 5). In detail, after three days culture in YPD liquid media, all strains with approximately the same amount of each strain were mixed together. Cells were centrifuged and suspended in water plus 7% DMSO and adjusted to an OD600 of 50. This pool of strains was then frozen at -80 °C. Drug screens were performed as described (6). Specifically, cells are grown in 0.7 ml YPD medium with drugs and keep shaking in GENios Tecan for 5 or 20 generations and sampled by a liquid handler. For each drug screen, a control treatment was included by growing cells in YPD media without drug. Drug concentration and generations used in each screen is listed in table 1. Each drug screen and microarray experiment was repeated three times.

DNA extraction, PCR and microarray experiment

DNA extraction, PCR and microarray were performed as described (5.) In detail, DNA from 1.5 ml culture (OD=1~2) was extracted using YeaStar genomic DNA kit (Zymo research). The barcodes in each DAmP strain were ampified using a pair of common primer flanking each barcode. For each sample, two PCRs are run. One for uptags (primers are 5’ end biotin labeled U1 and U2) and one for downtags (primers are 5’ end biotin labeled D1 and D2). Each PCR reaction contains 41 μl of high fidelity PCR SuperMix (Invitrogen), 8 μl of genomic DNA (~0.1 μg) and 1 μl of 50 uM Up and Down primer mix. PCR conditions were: 94°C, 1 min; 55°C, 1 min; 68°C, 2 min for 35 cycles; then 68°C, 10 min. For chip hybridization, chips were first washed by 90 ul of hybridization buffer (0.3 mM of MES-free acid monohydrate, 0.73 mM of MES sodium salt, 0.885M NaCI, 20 mM EDTA and 0.01% Tween 20, pH 6.5-6.7) by incubating at 42 °C for at least 10 min with rotation at 20 r.p.m. in the hybridization oven. Hybridization buffer were then replaced by 150 μl of hybridization buffer mix (75 μl of 2X hybridization buffer, 0.5 μl of B213 oligo (5’ biotin-CTG AAC GGT AGC ATC TTG AC-3’, 0.2fm/ul), 12 μl mixed oligos (12.5pm/ul), 3μl of 50x Denhart's solution, 30 μl of uptag PCR product and 30 μl of downtag PCR product, boiled for 2 min and set on ice for at least 2 min). A tough-spot was placed over each of the two gaskets to prevent evaporation. Hybridize for 16 hours at 42 °C rotating at the array at 20 r.p.m. After hybridization, the chips were washed with wash A (6x SSPE and 0.01% Tween 20) and wash B (3x SSPE and 0.01% Tween 20) in fluidics station (Affymetrix Gene Chip fluidics station 450) using the protocol “Genflex_TAG4_wash_protocol”. Finally, the chips were scanned at an emission wavelength of 560 nm.

References:

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2. Giaever, G. et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387-391 (2002).

3. Hillier, L.W. et al. Whole-genome sequencing and variant discovery in C. elegans. Nat Methods 5, 183-188 (2008).

4. Tong, A.H. et al. Systematic genetic analysis with ordered arrays of yeast deletion mutants. Science 294, 2364-2368. (2001).

5. Pierce, S.E., Davis, R.W., Nislow, C. & Giaever, G. Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures. Nature protocols 2, 2958-2974 (2007).

6. Giaever, G. et al. Chemogenomic profiling: identifying the functional interactions of small molecules in yeast. Proc Natl Acad Sci U S A 101, 793-798 (2004).