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Sample GSM7884349

Query DataSets for GSM7884349

Status

Public on Apr 24, 2024

Title

Library_induced_1mil_cells_G8_gate

Sample type

SRA

Source name

DHY211

Organism

Saccharomyces cerevisiae

Characteristics

cell line: DHY211

Treatment protocol

We treated cells with ß-estradiol for 4hrs before cell sorting

Extracted molecule

genomic DNA

Extraction protocol

We sorted 1e6/500k cells into each pool. We extracted genomic DNA from these cells with the Zymo YeaSTAR (#D2002) kit.
To ensure singular constructs per cell, we introduced our library into the URA3 locus of strain DHY211 (MATa, MKT1(30G,) RME1(INS-308A) TAO3(1493Q), CAT5(91M) MIP1(661T), SAL1+ HAP1+). Employing the established yeast transformation method (64), we subjected the transformation to 30 minutes at 30 °C followed by 60 minutes at 42 °C. To minimize potential PCR errors, we performed SalI and EcoRI digestion on the plasmid library, releasing the section encompassing the ACT1 promoter, the synthetic TF, and the KANMX marker. Simultaneously, PacI digestion was conducted to cleave plasmids devoid of an activation domain variant and barcode insert, thereby reducing the occurrence of transformants with inactive TFs. Directed integration into the URA3 locus was guided by 500 bp upstream homology spanning the URA3 and ACT1 promoters, along with a corresponding 500 bp downstream homology region spanning the TEF and URA3 terminators. These regions were PCR amplified from pMVS 295 (Strader 6161) and pMVS 296 (Strader 6768), a generous gift from Nick Moffy and Lucia Starder. Transformation utilized a molar ratio of 1:3 for linearized library to homology arms, with 28 μmol of linearized library per reaction. The transformed library was plated on YPD, followed by an overnight incubation at 30°C, and subsequent replica-plating onto freshly prepared SC G418 plates. Employing this process across 80 transformation reactions yielded an estimated >1,000,000 individual colonies. Subsequently, the transformants were collectively mated with an FY5 strain containing the reporter integrated into the uncertain ORF, YBR032w. Diploids were selected on YPD with G418 (200 µg/ml) and NAT (100 µg/ml) (strain MY436 YBR032w::P3_GFP NAT S288C), resulting in prototrophic diploids. These 110,000 yeast transformants were mated in batches, and prior to the final experiment, batches were pooled, and multiple aliquots were frozen.
beta-estradiol inducible library

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

non_TF_activation_domains_from_yeast_plant_1mil_G8

Data processing

For basecalling we used PEAR v0.9.11
After demultiplexing samples, we kept only the reads that contained a perfect match to a designed tile. For each set of 8 sorted samples, we performed two normalizations. We first normalized the reads by the total number of reads in each bin. Then, for each designed tile, we normalized across the 8 bins to calculate a relative abundance. We then converted relative abundances to an activity score for each tile by taking the dot product of the relative abundance with the median fluorescence value of each bin. This computation is a weighted average. Tiles with less than 10 reads were not included in the final dataset. Later, post hoc analysis suggested that tiles with at least 1000 reads were well measured.
Assembly: N/A

Submission date

Nov 06, 2023

Last update date

Apr 24, 2024

Contact name

Niklas Hummel

Organization name

Lawrence Berkeley National Laboratory

Street address

1 Cycloton Road

City

Berkeley