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Sample GSM8023801

Query DataSets for GSM8023801

Status

Public on Mar 21, 2024

Title

BLX assay timepoint 0

Sample type

SRA

Source name

cell

Organism

Saccharomyces cerevisiae

Characteristics

tissue: cell
cell line: Y1HGold (Takara Bio)
treatment: none

Treatment protocol

Geneticin was added to one lineage of each strain (LE9; 400 µg/ml, BLX; 500 µg/ml) starting from timepoint 0

Growth protocol

Competition assays were performed using a complete supplement mixture without histidine (Formedium) and yeast nitrogen base without amino acids (Formedium) media, supplemented with 2% glucose. To begin, the barcoded PAX6 variant library was transformed into two separate strains, LE9 and BLX, in two independent assays. The transformation process followed the protocol described in the Yeastmaker™ Yeast Transformation System 2 User Manual (Takara Bio), yielding approximately 2 x 106 and 7 x 106 transformants for LE9 and BLX strains, respectively. Transformed cells were then inoculated into 500 ml of liquid media and incubated at 30°C and 230 RPM for 12-24 hours until reaching an OD600 of approximately 1.0. Subsequently, approximately 7.5 x 108 cells were transferred to fresh media and grown for an additional 12 hours (T0). The T0 cultures were then split into two parallel lineages. All cultures were then maintained between an OD600 of 0.05 and 1.0 by passaging every 12 hours for a total of 36 hours (Timepoints 1-3).

Extracted molecule

other

Extraction protocol

Plasmid isolation was achieved using the protocol described by Fowler et al. (2014), which in summary used a freeze-thaw cycle and zymolyase cell wall digestion steps were coupled with Qiaprep spin miniprep kit (Qiagen) column purification
Samples from each timepoint were prepared for next-generation sequencing using a single limited-cycle PCR. Barcodes were amplified using primers possessing overhangs that contained sequencing primer annealing sequences, unique dual indexes, and P5/P7 Illumina adapter sequences (Table S1). 50 µl PCR reactions were performed for each timepoint using Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) with 20 to 50ng of template isolated plasmid DNA. Each reaction was then thermocycled using the following conditions: (1) initial denaturation at 98°C for 30 seconds, (2) incubation at 98°C for 8 seconds, (3) incubation at 69°C for 20 seconds, (4) incubation at 72°C for 8 seconds, (5) incubation at 72°C for 7 minutes. Steps 2 to 4 were repeated for 12 to 19 cycles (< 20). Multiple replicate 50 µl reactions were performed for each timepoint to ensure enough PCR product was generated. 2.5ul of Exo I nuclease (NEB) was then added directly to each ‘dirty’ PCR product and incubated at 37°C for 1 hour, followed by purification using QIAquick PCR Purification Kit (Qiagen). Samples were further purified by 2% E-Gel SizeSelect agarose gel (Invitrogen). The resulting products were purified using QIAquick PCR Purification Kit (Qiagen), after which all samples were pooled at equimolar amounts (quantified by QuBit [Invitrogen]) and then quantitated by Bioanalyzer (Agilent).

Library strategy

OTHER

Library source

other

Library selection

other

Instrument model

NextSeq 2000

Description

NextSeq 1000/2000 P2 Reagents (100 cycles) v3 Kit (#20046811) with custon rd1 primer and PhiX Control v3 spiked in to the sequencing library at a concentration of 10%
blx_0to03_g418.tsv

Data processing

For pax6_le9_assay, Data from the run was transferred from the NextSeq 550 instrument to a computer running Linux and a sample sheet was created to allow the generation of demultiplexed FASTQ files using Bcl2fastq2 v2.20.0.422, using adapter stringency 0.9.
For pax6_blx_assay, Basecall data was produced by the NextSeq 1000/2000 Control Software (Version 1.4.1.39716).
Following demultiplexing of the pax6_le9_assay and pax6_blx_assay, the regions flanking the 30 nt barcodes were trimmed using trimgalore then barcodes counted and variant fitness scores calculated using the Enrich2 tool (Rubin et al. 2017)
Assembly: NA
Supplementary files format and content: barcode counts calculated with the Enrich2 tool (Rubin et al. 2017)
Supplementary files format and content: Barcode:variant phasing was achieved using the alignparse Python package (Crawford et al. 2016)
Library strategy: Barcode amplicon sequencing

Submission date

Jan 18, 2024

Last update date

Mar 21, 2024

Contact name

Grzegorz Kudla

E-mail(s)

gkudla@gmail.com

Phone

+44 (0) 131 332 2471

Organization name

University of Edinburgh