Direct In-Sample Sequencing of the 3′ Transcriptome Expands the Capabilities of Optical Pooled Screens

  1. Daniel Honigfort
  2. Pedro Belda-Ferre
  3. David White
  4. Kousik Sundararajan
  5. Mariam Dawood
  6. Jake LeVieux
  7. Jacob Moreno
  8. Xiaodong Qi
  9. Kyle Metcalfe
  10. Duuluu Naranbat
  11. Andrew Altomare
  12. Connor Thompson
  13. Carlos Ruiz Perez
  14. Bryan Lajoie
  15. Hyukjin J. Kwon
  16. Pazinah Bhadha
  17. Tristin Rammel
  18. John Rabalais
  19. Matthew Kellinger
  20. Semyon Kruglyak
  21. Sinan Arslan
  22. Michael Previte
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Abstract

We present a platform that directly sequences single guide RNAs and endogenous 3′UTRs in fixed cells while simultaneously measuring protein abundance and cellular morphology. We demonstrate platform capability by performing optical pooled screening of CRISPR-perturbed lung cancer cells. This approach unites direct in-sample RNA sequencing with complementary phenotypic readouts, enabling comprehensive, scalable, and functional genomics analyses within a single experiment.

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