NextSeq 550 paired end sequencing; GSM3130435: egfp_unmod_1; Homo sapiens; RNA-Seq

egfp_unmod_1

Predicting the impact of cis-regulatory sequence on gene expression is a foundational challenge for biology. We combine polysome profiling of hundreds of thousands of randomized 5' UTRs with deep learning to build a predictive model that relates human 5' UTR sequence to translation. Together with a genetic algorithm, we use the model to engineer new 5? UTRs that accurately target specified levels of ribosome loading, providing the ability to tune sequences for optimal protein expression. We show that the same approach can be extended to chemically modified RNA, an important feature for applications in mRNA therapeutics and synthetic biology. We test 35,000 truncated human 5' UTRs and 3,577 naturally-occurring variants and show that the model accurately predicts ribosome loading of these sequences. Finally, we provide evidence of 47 SNVs associated with human diseases that cause a significant change in ribosome loading and thus a plausible molecular basis for disease. Overall design: Polysom profiling and sequencing was performed using a library of 300,000 randomized 5'' UTR 50-mers with eGFP used as the CDS. Three RNA chemistries were tested: unmodified, pseudouridine, and 1-methylpseudouridine. These were performed in duplicate (6 samples total). A designed library that includes human 5'' UTRs, SNVs, and sequences engineered with a genetic algorithm was used with the eGFP CDS (no duplicate). A second randomized library used mCherry as the CDS, also performed in duplicate.

Human 5' UTR design and variant effect prediction from a massively parallel translation assay

Submitted on 04-MAY-2018

Predicting the impact of cis-regulatory sequence on gene expression is a foundational challenge for biology. We combine polysome profiling of hundreds of thousands of randomized 5' UTRs with deep learning to build a predictive model that relates human 5' UTR sequence to translation. Together with a genetic algorithm, we use the model to engineer new 5? UTRs that accurately target specified levels of ribosome loading, providing the ability to tune sequences for optimal protein expression. We show that the same approach can be extended to chemically modified RNA, an important feature for applications in mRNA therapeutics and synthetic biology. We test 35,000 truncated human 5' UTRs and 3,577 naturally-occurring variants and show that the model accurately predicts ribosome loading of these sequences. Finally, we provide evidence of 47 SNVs associated with human diseases that cause a significant change in ribosome loading and thus a plausible molecular basis for disease. Overall design: Polysom profiling and sequencing was performed using a library of 300,000 randomized 5' UTR 50-mers with eGFP used as the CDS. Three RNA chemistries were tested: unmodified, pseudouridine, and 1-methylpseudouridine. These were performed in duplicate (6 samples total). A designed library that includes human 5' UTRs, SNVs, and sequences engineered with a genetic algorithm was used with the eGFP CDS (no duplicate). A second randomized library used mCherry as the CDS, also performed in duplicate.

Salt solution (10x): 100 mM NaCl, 100 mM MgCl2,100 mM Tris-HCl pH 7.5, and RNase-free water (Zuccotti P, Modelska A 2016). Wash buffer: 100 µg/ml cycloheximide in RNase-free DPBS (10 ml per plate). Lysis buffer: 1x salt solution, 1% of 20% Triton X-100, 1 mM DTT, 0.2 U/µl SUPERase-In (Thermo Fisher Scientific), 100 µg/ml cycloheximide. Wash buffer and lysis buffer were chilled throughout the protocol. After 12 hours of growth at 37 °C, cells were placed on ice and media was aspirated. Translating ribosomes were halted by adding 5 ml of wash buffer and were then placed at 37 °C for 5 minutes followed by aspiration on ice. Washed by adding 5 ml wash buffer and aspirating thoroughly. Added 300 µl of ice-cold lysis buffer, scraped cells, broke up cell clumps by pipetting ~5 times, and then placed suspended cells into a pre-chilled microcentrifuge tube. Let sit for 10 minutes on ice and then triturated by passing through a 25-G needle 10 times [1]. Spun at 16,000 x g for 5 minutes to pellet cell debris and nuclei. Saved supernatant and added 1.5 µl of 1 U/µl DNase I (final concentration of 0.005 U/µl) and placed on ice for 30 minutes. Either stored at -80 °C or proceeded directly to polysome profiling. For both the eGFP and mCherry library, randomized 50-mer oligos were Gibson assembled to serve as 5' UTRs for each CDS. To create the designed library, oligos were made via oligo synthesis array (CustomArray Inc.) and cloned into the eGFP vector.For all libraries, 25 nucleotides of defined sequence preceed the inserted 5' UTRs.

Human 5' UTR design and variant effect prediction from a massively parallel translation assay

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