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Sample GSM8023808 Query DataSets for GSM8023808
Status Public on Mar 21, 2024
Title PAX6-barcode phasing
Sample type SRA
Source name synthetic DNA
Organism synthetic construct
Characteristics tissue: synthetic DNA
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 Sequel II
Description a region of the plasmid containing the barcodes, PAX6, and GAL4 was excised from the vector followed by BluePippin purification and sequenced using PacBio Sequel II and 20 hours of movie collection time
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
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
Phone +44 (0) 131 332 2471
Organization name University of Edinburgh
Department MRC HGU
Street address Crewe Road
City Edinburgh
ZIP/Postal code EH4 2XU
Country United Kingdom
Platform ID GPL33815
Series (1)
GSE253580 Deep mutational scanning quantifies DNA binding and predicts clinical outcomes of PAX6 variants
BioSample SAMN39486644
SRA SRX23277859

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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