Positive Selection Screen #1

Aim: Identifying genes that when knocked down confer resistance to cytotoxic Compound X. Cytotoxic Compound X kills 95%-99% cells within 48 hours when administered at a 1 nM concentration.

shRNA library used: 55K HGW shRNA Library Module 1

Procedure:

Day 1. 5 × 107 cells transduced at MOI 0.5 (40% transduction efficiency expected)

Day 2. Media change

Day 4. Actual transduction efficiency checked by flow cytometry. Start of puromycin selection (1 µg/ml)

Day 7. End of puromycin selection

Day 8. Culture split 50:50 into 2 samples (>1 × 108 cells/sample)

  • Sample A: cytotoxic Compound X added at a 1 nM concentration
  • Sample B: untreated

Day 10. Both samples harvested for genomic DNA isolation and NGS of integrated barcodes. For the untreated sample, 400 µg of genomic DNA is used for PCR and NGS of barcodes. For the treated sample, all of the recovered genomic DNA is used.

  • shRNAs are evaluated based on barcode enrichment in Sample A vs. Sample B
  • Gene hits are identified based on evaluation of targeting shRNAs


Positive Selection Screen #2

Aim: Identifying genes required for the transactivation of Promoter X by Compound Y. GFP is expressed from Promoter X and accumulates in cells within 24 hours of stimulation by Compound Y at a 10 nM concentration.

shRNA library used: 55K HGW shRNA Library Module 1

Procedure:

Day 1. 5 × 107 cells transduced at MOI 0.5 (40% transduction efficiency expected)

Day 2. Media change

Day 4. Actual transduction efficiency checked by flow cytometry. Start of puromycin selection (1 µg/ml)

Day 7. End of puromycin selection

Day 8. Compound Y added at 10 nM concentration.

Day 9. 1 × 108 cells sorted by FACS into two samples:

  • Sample A: bottom 1% dimmest GFP cells
  • Sample B: top 50% brightest GFP cells

Both samples are harvested immediately after sorting for genomic DNA isolation and NGS of integrated barcodes. For the top 50% population, 400 µg of genomic DNA is used for barcode PCR and NGS. For the bottom 1% population, all of the recovered genomic DNA is used.

  • shRNAs are evaluated based on barcode enrichment in Sample A vs. Sample B
  • Gene hits are identified based on evaluation of targeting shRNAs

Last modified: Jan 10, 2019

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