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Sample GSM7900192

Query DataSets for GSM7900192

Status

Public on Apr 27, 2024

Title

80_FL_activity_block2_output2

Sample type

SRA

Source name

log phase culture

Organisms

Saccharomyces cerevisiae; Homo sapiens

Characteristics

tissue: log phase culture
cell line: BY4741
cell type: log phase culture

Treatment protocol

abundancePCA (High-resolution mapping of protein concentration reveals principles of proteome architecture and adaptation, Levy and Michnick, 2014), activity-dependent toxicity assay (A Combined Approach Reveals a Regulatory Mechanism Coupling Src’s Kinase Activity, Localization, and Phosphotransferase-Independent Functions, Ahler et al., 2019)

Growth protocol

BY4741 cells were transformed with the Src variant library cloned in the activity and abundance assay plasmids at a coverage of at least 20x and transferred to selective media for ~24h. The resulting cell population was transferred to input media (SC -ura-ade 2% glucose for abundance selections and SC -ura 2 % raffinose 0.1% glucose for activity selections) and grown overnight to an OD of at least 1.6. A small volume of cells was saved to inoculate outputs and the rest of cells were split in two, harvested by centrifugation, and frozen at -20C as input samples. The remainder of cells were then inoculated at an OD=0.05 in selective media (SC -ura-ade+MTX 2% glucose for abundance, and SC -ura 2% galactose 0.1% glucose for activity), and grown to an OD=1.6 at which point cells were harvested and stored as output samples as described for the inputs.

Extracted molecule

genomic DNA

Extraction protocol

Cell pellets (one for each experiment input/output replicate) were re-suspended in 1 mL of DNA extraction buffer (2% Triton-X, 1% SDS, 100mM NaCl, 10mM Tris-HCl pH8, 1mM EDTA pH8), and freeze-cracked by liquid nitrogen freezing followed by 62ºC incubation for 10 min in a water bath, twice. Subsequently, 1 mL of Phenol/Chloro/Isoamyl 25:24:1 (equilibrated in 10mM Tris-HCl, 1mM EDTA, pH8) was added, together with 1 g of acid-washed glass beads (Sigma Aldrich) and the samples were vortexed for 10 minutes. Samples were centrifuged at RT for 30 minutes at 4,000 rpm and the aqueous phase was transferred into new tubes. The same step was repeated twice. 0.1 mL of NaOAc 3M and 2.2 mL of pre-chilled absolute ethanol were added to the aqueous phase. The samples were gently mixed and incubated at -20ºC for 30 minutes. After that, they were centrifuged for 30 min at full speed at 4ºC to precipitate the DNA. The ethanol was removed and the DNA pellet was vacuum-dried. DNA pellets were resuspended in 0.6 mL TE 1X and treated with 5 uL of RNaseA (10mg/mL, Thermo Scientific) for 30 minutes at 37ºC. To desalt and concentrate the DNA solutions, QIAEX II Gel Extraction Kit was used (30 μL of QIAEX II beads). The samples were washed twice with PE buffer and eluted in 200 μL of 10 mM Tris-HCI buffer, pH 8.5.
Plasmid concentrations in the total DNA extract (that also contained yeast genomic DNA) were quantified by qPCR using the primer pair oGJJ152-oGJJ153, that binds to the ori region of the plasmids. We performed 2 consecutive PCR reactions for each sample. The first PCR (PCR1) is used to amplify the amplicons for sequencing, to add a part of the Illumina sequencing adaptors to the amplicon and to increase nucleotide complexity for the sequencing reaction by introducing frame-shift bases between the adapters and the sequencing region of interest. The second PCR (PCR2) is used to add the remainder of the Illumina adaptor and to add demultiplexing indexes (dual-indexed). The amplicon libraries were run on a 2% agarose gel for quality control and quantification, and were pooled by block and size selected on a 2% agarose gel. Bands were purified using QIAEX II Gel Extraction Kit (QIAGEN) and using 30uL of QIAEX II beads for each sample. The purified amplicons were subjected to Illumina 150bp paired-end NextSeq sequencing at the CRG Genomics Core Facility.
Amplicon-seq

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

NextSeq 2000

Data processing

DiMSum (Demultiplex with Cutadapt)
DiMSum (QC raw reads with FastQC)
DiMSum (Trim reads with Cutadapt)
DiMSum (Process and analyse variants with DiMSum)
Assembly: NA
Supplementary files format and content: Fitness scores table: Tabular format; contains the raw counts and dimsum fitness estimates per replicate and aggregated and their corresponding errors.
Supplementary files format and content: Weight tables: Tabular format; contains the MoCHI inferred 'folding' and 'activity' energies

Submission date

Nov 14, 2023

Last update date

Apr 27, 2024

Contact name

Antoni Beltran

E-mail(s)

toni.beltran@crg.eu

Organization name

CRG Barcelona

Street address

Dr Aiguader, 88