Negative Selection Screen #1

Aim: Identifying shRNAs that are differentially cytotoxic in Cell Line A vs. Cell Line B.

shRNA library used: 55K HGW shRNA Library Module 1

Cell Lines A and B each have a doubling time of ~36 hours.

Procedure:

Day 1. 5 × 107 cells per cell line 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, ~3 × 108 cells/sample. In some cases, it can be appropriate to continue puromycin selection throughout the experiment. All cells from each sample are pooled together and re-plated at 6 × 107 cells/sample.

Day 10. All cells from each sample are pooled together and re-plated at 6 × 107 cells/sample.

Day 13. All cells from each sample are harvested for genomic DNA isolation and NGS of integrated barcodes. 400 µg genomic DNA per sample is used for PCR and NGS of barcodes.

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


Negative Selection Screen #2

Aim: Identifying shRNAs that synergize with cytotoxic effect of Compound X in Cell Line A. Compound X has IC50 = 1 nM when administered continuously for 7 days.

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, ~3 × 108 cells. In some cases, it can be appropriate to continue puromycin selection throughout the experiment. Cells are pooled and re-plated into 2 separate samples, 6 × 107 cells/sample. Sample A is treated with 1 nM Compound X, and Sample B is mock-treated.

Day 10. All cells from each sample are pooled and re-plated at 6 × 107 cells/sample. Sample A is still treated with 1 nM Compound X, and Sample B is still mock-treated.

Day 13. All cells from each sample are harvested for genomic DNA isolation and NGS of integrated barcodes. 400 µg genomic DNA per sample is used for barcode amplification and NGS.

  • shRNAs are evaluated based on barcode depletion 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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