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Sample GSM2643724

Query DataSets for GSM2643724

Status

Public on Mar 06, 2018

Title

OUTPUT_12

Sample type

SRA

Source name

yeast culture

Organism

Saccharomyces cerevisiae

Characteristics

strain: BY4742 hsx1::natMX4
growth protocol: SC -HIS 1M NaCl at 37ºC

Growth protocol

Yeast was grown in synthetic complete media -HIS at 30ºC (for input samples) and SC -HIS 1M NaCl at 37ºC (output samples). Cells were harvest, centrifuged and pellets saved at -20ºC for DNA extraction

Extracted molecule

genomic DNA

Extraction protocol

Cell pellets were resupended in 1.5 mL extraction buffer (2% Triton-X, 1% SDS, 100mM NaCl, 10mM Tris-HCl pH8, 1mM EDTA pH8), frozen by dry ice-ethanol bath and incubated at 62ºC water bath twice. Subsequently, 1.5mL of Phenol/Chloro/Isoamyl 25:24:1 (equilibrated in 10mM Tris-HCl, 1mM EDTA, pH8) was added, together with 1.5 g of glass beads and the samples were vortexed for 10 min. Samples were centrifuged at RT for 30 min at 4,000 rpm to phase separate and the aqueous phase was transferred into new tubes. The same step was repeated twice. 1:10V of NaOAc 3M and 2.2V of cold ethanol 100% were added to the aqueous phase. The mix was incubated at -20ºC for 30 min and after that, centrifuged for 30 min at full speed at 4ºC to precipitate the DNA. The ethanol was removed and the DNA pellet allowed to dry overnight at RT. DNA pellets were resuspended in 900 µL TE 1X and treated with RNAseA (10 mg/mL, Thermo Scientific) for 30 min at 37ºC. To desalt and concentrate the DNA solution, QIAEX II Gel Extraction Kit was used (75 µL of QIagen beads). Finally the sample was washed 3 times with PE and eluted twice in 375 10 mM Tris·Cl, pH 8.5
A 2-step PCR using high fidelity Q5 Hot Start High-Fidelity DNA Polymerase (NEB) was used to amplify the input and output libraries for sequencing. In each sample, ~30 million plasmid molecules were amplified for 10 cycles using oligos with overhang homology to Illumina sequencing adapters (Supplementary Table 3). The first PCR reaction was performed independently for each of the 8 samples. The samples were then treated with ExoSAP (Affymetrix), concentrated by bead purification with a QIAEX II kit. The entire first PCR reaction was used for the second PCR reaction (15 cycles), where the rest of the Illumina adapter was added multiplexed with different indexes. The DNA concentration of each individual second PCR was quantified by fluorometric quantitation (Quant-iT™ PicoGreen® dsDNA Assay Kit) and pooled together in an equimolar ratio. Finally, the pooled sequencing library was run on a gel purified and concentrated (QIAEX II Gel Extraction Kit) and subjected to 125bp paired-end sequencing on an Illumina HiSeq 2500v5 sequencer.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

Sample 4
DNA-seq of PCR amplicon from plasmid library

Data processing

Read trimming (with cutadapt)
Paired end read merging (PEAR)
Base-calling (custom python scripts)
Supplementary_files_format_and_content: Tabular format. Headers: seq=tRNA genotype; num_vars=Number of mutations compared to S. cerevisiae WT genotype; pos_vars=Position of the mutation/s; ntd_vars=Nucleotide substitutions; ntd_wt=WT nucleotides. Columns with "reads" in the headers correspond to raw input reads (after filtering, see Methods); fitness=ln(fitness) values relative to the S. Cerevisiae WT genotype, SE=standard error of the fitness estimate

Submission date

May 30, 2017

Last update date

May 15, 2019

Contact name

Julia Domingo

E-mail(s)

julia.domingo.espinos@gmail.com

Organization name

New York Genome Center

Lab

Lappalainen Lab