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Sample GSM8229583

Query DataSets for GSM8229583

Status

Public on Apr 25, 2024

Title

cript_n_input_biolrep2

Sample type

SRA

Source name

lab strain BY4741

Organism

Saccharomyces cerevisiae

Characteristics

cell type: yeast cells
strain: lab strain BY4741
variant: cript_n
group: input

Growth protocol

For each of the libraries of variants (cript_c, cript_n, pdz3cript [in 2 blocks, B1 and B2] and cript2xa3), each of the growth competitions were performed following transformation of yeast with each library in biological triplicates. After the first cycle of post-transformation plasmid selection, a second plasmid selection cycle (input) was performed by inoculating SD -URA/ADE at a starting OD600nm = 0.1 with the saturated culture. Cells were grown for 5 generations at 30ºC under constant agitation at 200 rpm. This allowed the pool of mutants to be amplified and enter the exponential growth phase. The competition cycle (output) was then started by inoculating cells from the input cycle into the competition media (SD -URA/ADE + 200 ug/mL Methotrexate) so that the starting OD600nm was 0.05. For that, the adequate volume of cells were collected, centrifuged at 3,000 rpm for 5 minutes and resuspended in the pre-warmed output media. Meanwhile, each input replicate culture was splitted in two and harvested by centrifugation for 5 min at 5,000g at 4ºC. Yeast cells were washed with water, pelleted and stored at -20ºC for later DNA extraction. After ~5 generations of competition cycle, each output replicate culture was splitted into two and harvested by centrifugation for 5 min at 5,000g at 4ºC, washed twice with water and pelleted to be stored at -20ºC

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), frozen by dry ice-ethanol bath and incubated at 62ºC 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. They were then centrifuged for 30 min at full speed at 4ºC to precipitate the DNA. The ethanol was removed and the DNA pellet was allowed to dry overnight at RT. 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 twice by 125 μL of 10 mM Tris-HCI buffer, pH 8.5 and then combined two elution. Finally, 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 (PCR1 and PCR2). In PCR1, we added frameshifting oligonucleotides and amplified the regions of interest (amplicon with constant region and mutated region) from the extracted DNA with 5 cycles. In PCR2 we added Illumina sequencing barcodes with PCR using the minimum number of cycles necessary to reach amplification plateau for each sample based on a qPCR run. The amplicon library pools were run on a 2% agarose gel and 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.
PCR amplicon

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

cript_n_sub.csv

Data processing

DiMSum (Demultiplex with Cutadapt)
DiMSum (QC raw reads with FastQC)
DiMSum (Trim reads with Cutadapt)
DiMSum (Process and analyse variants with DiMSum)
Raw read files LTZ020_lib_04737AAD_RS2_GTCGATGC_R1_001.fastq.gz and LTZ020_lib_04737AAD_RS2_GTCGATGC_R2_001.fastq.gz were also used as technical replicates for cript2xa3_cis_input_biolrep1. Raw read files LTZ019_lib_04736AAD_RS2_TCAGATAC_R1_001.fastq.gz and LTZ019_lib_04736AAD_RS2_TCAGATAC_R2_001.fastq.gz were also used as technical replicates for cript2xa3_cis_input_biolrep2. These shared the same barcode with other samples (pdz3cript_b1_wt_ctrl and pdz3cript_b2_input_biolrep1) but contain different amplicons and therefore were further de-mutiplexed in data processing based on the specific designed sequences for each experiment.
Assembly: GRCh38
Supplementary files format and content: Tabular format with headers: aa_seq=amino acid sequence, WT=True if it is the wild type sequence, fitness=Average fitness/binding score of all replicates calculated by DiMSuM, sigma=standard error of the fitness measurement calculated by DiMSuM
Supplementary files format and content: For the processed files, the fitness estimates for the pdz3cript library were first normalized between block1 and block2 using a linear model between STOPs and synonymous substitutions in each block. Since all libraries shared common variants with the pdz3cript library and were strongly correlated (Pearson's r > 0.9), the common variants were used to linearly scale the fitness scores and sigma (error) associated with each variant.

Submission date

Apr 24, 2024

Last update date

Apr 25, 2024

Contact name

Taraneh Zarin

E-mail(s)

taraneh.zarin@gmail.com

Organization name

Centre for Genomic Regulation

Lab

Ben Lehner