biostudies-arrayexpressMartin MiklHomo sapiensultraplex (https://github.com/ulelab/ultraplex), pear (v0.9.11), STAR (v2.7.10a)samtools (v1.4), BCFtools (v1.10.2-GCC-8.3.0)https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16588The human protein Shiftless was recently identified as a broad-spectrum, but relatively low efficacy inhibitor of programmed ribosomal frameshifting (PRF). Here, we report results from an integrated high-throughput screening system that enables quantitative assessment of PRF inhibition by large libraries of protein variants. Using this platform, we performed deep mutagenesis of Shiftless and measured the activity of over 5,000 variants in a cell-based dual-fluorescence reporter assay with NGS-based readout.biostudies-arrayexpressSequencing - Sequencing was performed on one lane of a NovaSeq X plus with 2x 150 cycles (Admera Health, New Jersey, USA).Growth Protocol - HEK293 cells were grown in DMEM+10%FBS+PenStrep at 37 degrees C and 5% CO2.Nucleic Acid Extraction - Genomic DNA was isolated using Monarch gDNA purification kit (NEB) according to the manufacturer’s protocol.Sample Collection - To sort cells based on their GFP/mCherry ratio, we defined four diagonal gates based on GFP to mCherry fluorescence intensity (as shown in Figure 2B) and sorted approximately 8 million cells into four bins using a Sony MA-900 sorter. Sorted cells were collected in full medium, transferred to 15 mL tubes, collected by centrifugation, resuspended in fresh medium and transferred to 6 well plates for growing. After we obtained at least 1 million cells in the bins with the lowest number of sorted cells (1 and 4), cells were collected for genomic DNA isolation.Sample Treatment - To generate pools of cells with genomically integrated plasmid libraries, 6 million HEK293 cells were transfected with a 20:1 mix of library plasmid and Cas9/sgRNA plasmid targeting the AAVS1 locus (PX459 (Addgene) with AAVS1-specific sgRNA using Polyjet (SignaGen). 6 hours after transfection, medium was changed to DMEM+10%FBS+PenStrep and 24 hours after transfection, medium was again replaced DMEM+10%FBS+PenStrep supplemented with 2 μg/ml puromycin. Cells were grown in the presence of 2 μg/ml puromcyin for 2 weeks until a uniform mCherry-positive population was reached.Library Construction - For library preparation, reporter genomic DNA was PCR amplified using reporter specific forward (g404, g406, g407) and reverse (n250) primers and introducing partial Illumina Read1 and Read2 sequences, respectively. Three PCRs with different forward primers (g404, g406, g407) were performed on each gDNA sample, constituting technical replicates accounting for any sampling bias. 20 ul Kapa hifi ready mix, 1.5 ul 10 uM primer reporter-specific forward primer (adding the Illumina TruSeq Read 2 sequence) and 1.5 ul reverse primer (adding the Illumina TruSeq Read 1), 200 ng gDNA, water to 40 ul (3 reactions per sample). 98 deg 3 min, 15 cycles: 98 deg 20 sec, 65 deg 15 sec, 72 deg 20 sec; 72 deg 1 min. After cleaning up the reaction with 1x Ampure beads (Beckman-Coulter) Illumina sequencing adaptors (NEBNext Multiplex Oligos for Illumina, NEB #E7600S) were added in a second PCR reaction: PCR program: 98 deg 3 min, 14 cycles: 98 deg 20 sec, 65 deg 15 sec, 72 deg 20 sec; 72 deg 1 min. The reactions were cleaned up with Ampure beads (0.75 x) and the size of the product was verified using Tapestation (Agilent High Sensitivity D1000 ScreenTape).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - Before mapping, Illumina sequencing reads were demultiplexed using ultraplex (https://github.com/ulelab/ultraplex) according to the replicate-specific barcodes incorporated in the first PCR reaction (see above). Paired forward and reverse reads, which by design overlapped, were merged using pear (v0.9.11). Merged reads were then mapped against the SHFL reference sequence as a genome using STAR (v2.7.10a) using the following commands: STAR --runThreadN 20 --runMode genomeGenerate --genomeDir ~/resources/shiftless --genomeFastaFiles ~/resources/shiftless/Shiftless_wildtype.fasta.txt --sjdbGTFfile ~/resources/shiftless/shfl_reference.gtf --sjdbOverhang 100 --sjdbGTFtagExonParentTranscript Parent --sjdbGTFfeatureExon gene STAR --runMode alignReads --genomeDir ~/resources/shiftless --readFilesIn samplename_pairedreads.fastq --outSAMtype BAM Unsorted --outFileNamePrefix samplenameData Transformation - The mapped bam file was sorted using samtools sort (v1.4) and variant calling was performed using BCFtools (v1.10.2-GCC-8.3.0) using the mpileup function and the following command: bcftools mpileup -f Shiftless_wildtype.fasta.txt sorted.bam --max-depth 100000000 -a AD -O vcf -o outfile.vcf The resulting vcf file (provided as processed data files) were the starting point for all position-based downstream computational analyses.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsSony MA-900 cell sorterIllumina NovaSeq XDNA-seqHomo sapiensMartin MiklfalseHigh-throughput functional testing of Shiftless variantsThe human protein Shiftless was recently identified as a broad-spectrum, but relatively low efficacy inhibitor of programmed ribosomal frameshifting (PRF). Here, we report results from an integrated high-throughput screening system that enables quantitative assessment of PRF inhibition by large libraries of protein variants. Using this platform, we performed deep mutagenesis of Shiftless and measured the activity of over 5,000 variants in a cell-based dual-fluorescence reporter assay with NGS-based readout.2026-08-06T00:00:00Z2026-08-06T13:37:45.779Z2026-01-28T14:37:49.429ZE-MTAB-16588ERP188262EFO_0002944EFO_0004170EFO_0003789EFO_0002693EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969