Perturb-Seq scRNA-seq for Alternative Promote Usage Sites
Gene expression (GEX) and sgRNA libraries were prepared following 10X Genomics Chromium Single Cell 5' Kit User Guide v2 (Dual Index) with Feature Barcode technology for CRISPR Screening (CG000510 Rev B).  Media was replaced 24 hours after transfection, and 48 hours later, supernatants containing viruses were collected by centrifugation and filtration through a 0.45-μm PVDF filter (Millex Millipore - 0890). The viruses were either used immediately or stored in aliquots at −80 °C. After 6 days, the cells were harvested for total RNA extraction using the RNeasy Mini Plus Kit (74134 - Qiagen) according to the manufacturer's instructions. cDNA was synthesized using RevertAid RT Reverse Transcription kit (K1691 - Thermo Fisher Scientific) following the manufacturer's protocol. By the 8th day post-transduction, more than 70% of the MCF-7 KRAB dCas9 population expressed the library (BFP+), and a final cell sorting (Aria II Cell Sorter - BD Biosciences) was conducted prior single-cell direct capture Perturb-Seq. PromCRISPRi cells with more than 90% viability were prepared as single-cell suspension and loaded into droplet emulsions to two lanes of Chromium Single Cell Chip K, aiming to recover ~20,000 cells per GEM group = 40,000 in total.