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Sample GSM5724749

Query DataSets for GSM5724749

Status

Public on Dec 17, 2021

Title

Sample 5: Src_CD_2

Sample type

SRA

Source name

Synthetic sequence

Organism

Saccharomyces cerevisiae

Characteristics

strain: Green Monster BY4741
treatment: Src Inhibitor: 1,2,4
treatment concentration: 1: 8000 nM, 2: 2000 nM, 4: 800 nM
molecule: Plasmid DNA

Treatment protocol

S. cerevisiae harboring Src variant library were grown in 2% galactose containing media to induce Src expression and in varying concentrations of indicated Src kinase inhibitor. Library was grown and 4 OD unit timepoints were harvested throughout growth. The indicated concentration of Src inhibitor was added at the same time Src kinase expression was induced by galactose.

Growth protocol

TOP10 E.coli were grown in LB supplemented with 100 ug/mL Ampicillin. BY4741 ∆PDR5 or BY4741 Green Monster S. cerevisiae were grown in C-Leu media supplemented with 2% galactose.

Extracted molecule

other

Extraction protocol

Harvested cells (4 OD units worth) at each sampled time point were pelleted and stored at -20ºC. Plasmid DNA was extracted using Zymogen Yeast Miniprep Kit I following supplied protocol.
Extracted barcodes were amplifed and sample indices appended using 2x KAPA2G Robust HotStart ReadyMix with Thermocycler conditions of: Initial denaturation at 95ºC for 3 min, followed by 17 cycles of 95ºC for 15s, 60ºC for 15s, and 72ºC for 15s. Amplicons were quantified using KAPA Library Quantification Kit.
DNA amplicon sequencing using custom primers

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

Targeted barcode amplification from extracted plasmid DNA
Src_1_8000_Score.csv, Src_2_2000_Score.csv, Src_4_800_Score.csv

Data processing

Sample 1 was sequenced on MiSeq (Illumina). Samples 2-5 were sequenced on NextSeq 500 (Illumina).
All reads were processed using bcl2fastq v.2.16 and ea-utils v.1.1.2-537.
Sample 1: Custom scripts were used to process these reads. R2 files were trimmed to 230bp.
Samples 2-5: Functional scores were calculated using Enrich2 v1.2 software and Src_Barcode_Map.txt.
Genome_build: NA
Supplementary_files_format_and_content: Src_Barcode_Map.txt provides the linkage between the barcode sequence (first column) and the full length variant sequence of Src's catalytic domain (second column). This document was generated through the following steps: 1)Custom subassembly scripts to link barcode sequence with full length catalytic domain sequence. 2) Custom subassembly filtering script for high quality reads. 3) Filtering out WT barcodes with aberrant activity score using 2x Std. Dev. of WT barcode mean activity score. 4) Spike in of 6 barcode sequences of control Src variants (3 each for K298M and T341I).
Supplementary_files_format_and_content: Src_Das_25_Score.csv, Src_DAS_100_Score.csv, Src_1_8000_Score.csv, Src_2_2000_Score.csv, and Src_4_800_Score.csv are the output of Enrich2 v1.2. The inputs for Enrich2 v1.2 were the corresponding "Raw Files" (Input, A, B, and C) that share the same Src Inhibitor and concentration. Variants are denoted by their amino acid level changes and each has a calculated functional score from Enrich2.

Submission date

Dec 08, 2021

Last update date

Dec 17, 2021

Contact name

Douglas Fowler

E-mail(s)

dfowler@uw.edu

Organization name

University of Washington

Department

Genome Sciences