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| Status |
Public on Aug 26, 2020 |
| Title |
S. pombe Sfp1 mutant expression data [252061310018_2_4_Spom_T60_SFP1] |
| Sample type |
RNA |
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| Source name |
T60_SFP1
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype/varation: S. pombe Sfp1 mutant
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| Treatment protocol |
On day 1, cells were grown in BMW with growth saturation times as follows: S. cerevisiae, N. castellii, C. glabrata: ~24 hours, K. lactis: ~48 hours, S. pombe: ~36 hours. On day 2, 50 ml cultures, inoculated at 1x106 cells/ml from the overnights were grown in BMW until glucose reached zero, in the New Brunswick shaker. Glucose levels were measured throughout growth, but more frequently as they were reaching zero, using the YSI 2700 Biochemistry Analyzer. When glucose levels reached zero, cells were counted using the Cellometer Auto M10, and enough cells were removed to inoculate 300 ml of BMW at 1x107 cells/ml. Samples were collected at 30 minutes, 60 minutes, 90 minutes as previously described (Thompson et al., 2013). Briefly, 50-ml conical tubes filled with the appropriate amount of 100% methanol to produce a 60/40 mixture once the sample is added. The methanol-filled tubes were stored at −80°C until ready for use. During sample collection tubes were placed in a rack in a dry-ice ethanol bath kept at approximately −40°C. Once the sample was added to the methanol, the methanol and media were separated from the cells by centrifugation and poured off. The conicals containing a cell pellet were then stored at −80°C until processed for permanent storage. For permanent storage, the cell pellets were then washed in 5 ml of nuclease-free water and spun for 5 min at 3700 rpm at 4°C. The supernatant was discarded and the pellet re-suspended in 2 ml of RNA later (Invitrogen) and transferred to 2 ml Sarstadt tubes for storage. The samples were left at 4°C for 24 hr before being moved to a −80°C freezer.
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| Growth protocol |
We used the following strains for each species: S. cerevisiae RM11-1a and BY4741, C. glabrata CBS 138, N. castellii 4310 (MATa ho::loxp), K. lactis Say 509 (MATa ade2 leu2 metA1 trp1 uraA1 nej1::LEU2, and S. pombe SPY73h+. All species were grown in the following rich medium (termed BMW): yeast extract (1.5%), peptone (1%), dextrose (2%), SC amino acid mix (Sunrise Science) 2 g/l, adenine 100 mg/l, tryptophan 100 mg/l, uracil 100 mg/l. The medium was chosen to minimize cross-species variation in growth. All strains were grown at 30°C except for N. castellii, which was grown at 25°C. Cultures were grown exactly as described above. SFP1 was deleted from haploid strains of each specie using homologous recombination. Parent and deletion strains are listed in Table S1, and the primers used in Table S2 of the associated paper for this dataset. For S. cerevisiae (RM11-1a), SFP1 was deleted with URA3. The plasmid, 106,pRS406 (URA3) was used to create the transformation fragment. URA3 was amplified from the vector with primers containing ~60 base pairs of homology to the up- and downstream sequences to SFP1. Phusion enzyme (NEB) was used with standard protocol and a gradient PCR was done to find the optimal annealing temperature. PCR fragments were pooled and cleaned up with a phenol/chloroform extraction. Transformation was done using a standard S. cerevisiae electroporation protocol. Correct integration was checked by PCR of genomic DNA isolated from deletion candidates using sequences that were up- and downstream of the gene and internal to the drug cassette. Negative PCR controls included the PCR for the ORF and PCR running from the ORF to the up and downstream locations. Positive PCR controls included the same PCR reactions on the genomic DNA of the parent strain. S. cerevisiae (BY4741) was obtained from Open Biosystems (5312). For N. castellii, C. glabrata, K. lactis and S. pombe, the transformation fragment was created using a fusion PCR method as described by Szewczyk et al.(Szewczyk et al., 2007) The Pringle primers were used to amplify the drug cassette of the NAT1MX4 (pAG25) and pFA6a-KANMX6 vectors (Wach et al., 1994). The P1/P3 and P4/P6 primer combinations were used to amplify the up and downstream sequences of SFP1 and also contained homology to the drug cassette. The P2/P5 primers were used to fuse the three fragments together in a single PCR reaction. The basic PCR cycling parameters used were as previously described (Wach et al., 1994), except that PCRs were done using Phusion enzyme (NEB) and temperatures were adjusted accordingly. Optimal PCR cycling conditions for each species was determined using a gradient PCR. PCR fragments were pooled and cleaned up with a phenol/chloroform extraction. Transformation was done using a protocol adapted from Kooistra et al., 2004 (Kooistra, Hooykaas and Steensma, 2004). Correct integration was checked by PCR of genomic DNA isolated from deletion candidates using sequences that were up and downstream of the gene (P1 and P6 primers) and internal to the drug cassette (Pringle internal check)., For S. paradoxus, the drug cassette was amplified out of pFA6a-3HA-KANMX6 vector with ultramer primers of 200 bp length, designed to amplify this region and also to add ~180 bp of homology to the upstream and downstream regions of the S. paradoxus SFP1 ORF. Additional PCRs were carried out to add ~1kb of homology up and downstream of the SFP1 ORF and then fusion PCR was carried out as described above. Negative PCR controls included the PCR for the ORF and also PCR running from the ORF to the up and downstream locations. Positive PCR controls included doing all of the same PCR reactions on the genomic DNA of the parent strains.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction and microarray hybridization were completed as described by Thompson et al.(Thompson et al., 2013): RNA preparation and labeling Total RNA was isolated using the RNeasy Midi or Mini Kits (Qiagen, Valencia, CA) according to the provided instructions for mechanical lysis.
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| Label |
Cy5
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| Label protocol |
Samples were quality controlled with the RNA 6000 Nano ll kit of the Bioanalyzer 2100 (Agilent, Palo Alto, CA). cDNA samples were labeled with either Cy3 or Cy5 using a modification of the protocol developed by Joe DeRisi (University of California at San Francisco) and Rosetta Inpharmatics that can be obtained at http://www.microarrays.org (Wapinski et al., 2010).
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| Hybridization protocol |
For each time point, either two or three biological replicates were hybridized. We used two-color Agilent 55- or 60-mer oligo-arrays in the 4 × 44 K or 8 × 15 K format for the S. cerevisiae strain (commercial array; four to five probes per target gene) or the custom 8 × 15 K format for all other species (two probes per target gene)
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| Scan protocol |
After hybridization and washing per the manufacturer’s instructions, arrays were scanned using an Agilent scanner and analyzed with Agilent’s Feature Extraction software (release 10.5.1.).
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| Description |
252061310018_201104071055_S01_GE2_105_Dec08_2_4.txt
S. pombe Sfp1 mutant expression data
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| Data processing |
From each set of data in the raw data the ProbeName, SystematicName, gMeanSignal, rMeanSignal, gBGMeanSignal, rBGMeanSignal features were selected for each DATA entry.
To define a value for each probe in the attached matrices, the values for gMeanSignal, rMeanSignal, gBGMeanSignal, rBGMeanSignal features were first averaged for the multiple entries for each individual probe. Then, for each probe in each data set the averaged values were background-subtracted, log-transformed. Each value in the species matrices corresponds to log(<gMeanSignal>-<gBGMeanSignal>+1) or log(<rMeanSignal>-<rBGMeanSignal>+1) for the Cy3 and Cy5 data, respectively. In cases where <gBGMeanSignal> < <gMeanSignal> or <rBGMeanSignal> < <rMeanSignal>, 0 was inserted. So it is these averaged and transformed values that appear in the attached matrices for each probe in each species dataset. For interpretability the systematic gene name mapped to each of the probes is given for each species data set.
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| Submission date |
Sep 07, 2018 |
| Last update date |
Jan 11, 2022 |
| Contact name |
Carl G de Boer |
| Organization name |
The Broad Institute
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| Lab |
Aviv Regev
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| Street address |
415 Main St
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL15300 |
| Series (1) |
| GSE119645 |
A comparative gene expression data set for functional Sfp1 knockdown mutants and wild type strains for five yeast Species in the LOG growth phase |
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| Supplementary data files not provided |
| Processed data are available on Series record |
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