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Sample GSM8316073

Query DataSets for GSM8316073

Status

Public on Jul 26, 2024

Title

C_terminal_Ab42_double_mutants_output2

Sample type

SRA

Source name

single cell eukaryotic microorganism

Organism

Saccharomyces cerevisiae

Characteristics

cell type: single cell eukaryotic microorganism
genotype: [psi-pin-] (MATa ade1-14 his3 leu2-3,112 lys2 trp1 ura3-52)
treatment: protein expression

Growth protocol

For each Aβ42 plasmid library, yeast cells were transformed in 3 biological replicas (1,2,3), with a minimum of 3, 2.2, 0.99 and 0.43 million transformants for each of the replicate of N_terminal_Ab42_double_mutants, C_terminal_Ab42_double_mutants, Combinatorial_1 and Combinatorial_2 libraries, respectively. Once the culture reached the exponential phase, cells were resuspended in 100-1000mL protein inducing medium (-URA, 20% glucose, 100uM Cu2SO4) at OD600=0.05. Selection was performed for each biological replica. After 24h, 25-50ml of cells were harvested for input pellets and a minimum of 75, 12, 230 and 1.5 million cells / replica (for N_terminal_Ab42_double_mutants, C_terminal_Ab42_double_mutants, Combinatorial_1 and Combinatorial_2 libraries, respectively) were plated on -URA-ADE selection medium plates (145cm2, Nunc, Thermo Scientific) and incubated for 6-7 days at 30C. The number of cells plated ensures a minimum coverage of 10 times each variant in the library. Finally, colonies were scraped off the plates and harvested for output pellets. Inputs and output pellets were stored at -20C and later treated for DNA extraction.

Extracted molecule

other

Extraction protocol

Input and output pellets (3 biological replicates each, 6 tubes in total) were resuspended in 2ml extraction buffer (2% Triton-X, 1% SDS, 100 mM NaCl, 10 mM Tris pH 8, 1 mM EDTA pH 8) and subjected to two cycles of freezing-thawing in an ethanol-dry ice bath and water bath at 62C for 10min each. 1.5ml of phenol:chloroform:isoamyl 25:24:1 was then added to the pellets and vortexed for 10min. The aqueous phase was recovered by means of 30min centrifugation at 3000 rpm. DNA was precipitated with 1:10 V NaOAc 3M and 2.2 V of 99% cold ethanol and incubated at -20C for 2h. After centrifugation, pellets were dried overnight at RT. The next day, pellets were resuspended in TE 1X buffer (10mM Tris pH8, 1mM EDTA pH8) and treated with 10ul RNase A (Thermo Scientific) for 1h at 37C. DNA was purified using a silica beads extraction kit (QIAEX II Gel Extraction Kit, Qiagen).
PCR amplification in two steps using Q5 High-Fidelity DNA Polymerase (NEB). In step 1, 50 (for Combinatorial_2) or 100 (for N_terminal_Ab42_double_mutants, C_terminal_Ab42_double_mutants and Combinatorial_1 libraries) million plasmids were amplified for 15 cycles using frame-shifted adaptor primers with a partial homology to standard Illumina sequencing primers. PCR products from the first step were used as templates in the second PCR step, where indexed Illumina primers were used for a 10 cycles amplification. The three input samples were pooled together equimolarly, and the same for the three output samples. The two final pools were purified from a 2% agarose gel using a silica beads extraction kit (QIAEX II Gel Extraction Kit, Qiagen).

Library strategy

OTHER

Library source

other

Library selection

other

Instrument model

Illumina HiSeq 2500

Description

PCR DNA amplicon from plasmid library

Data processing

FastQ files from paired-end sequencing of each Aß42 library before (‘input’) and after selection (‘output’) were processed using the DiMSum pipeline (https://github.com/lehner-lab/DiMSum)
Assembly: NA
Supplementary files format and content: Spreadsheet file with columns indicating aa_seq = amino acid sequence of variant, aa_seq_full = full amino acid sequence of AB42 containing the variant (ready to feed in MoCHI), dataset = dataset label (N_terminal_Ab42_double_mutants, C_terminal_Ab42_double_mutants, Combinatorial_1, Combinatorial_2), mean_count= Mean read counts of the input replicates, fitness= Merged fitness, sigma = Merged error over fitness, fAD_category = Associated pathogenicity (non-fAD, fAD).
Library strategy: DNA-seq

Submission date

Jun 10, 2024

Last update date

Jul 26, 2024

Contact name

Mireia Seuma

E-mail(s)

[email protected]

Organization name

Institute for Bioengineering of Catalonia

Street address

Carrer de Baldiri Reixac, 10, 12