GSM6752591
After trypsinization, cells in each 10cm dish were collected into a 15ml falcon tube and kept on ice. Cells were spun down at 300xg for 5 minutes (4 °C) and washed once in 3ml ice-cold PBS. Cells were fixed with 5ml ice-cold 4% PFA in PBS for 15 minutes on ice. PFA was then quenched by adding 250ul 2.5M Glycine, and cells were pelleted at 500xg for 5 minutes (4 °C). Fixed cells were washed once with 1ml PBSR (PBS, 0.1% SUPERase In, and 10mM dithiothreitol (DTT)), and were then resuspended, permeabilized, and further fixed in 1ml PBSR-triton-BS3 (PBS, 0.1% SUPERase In, 0.2% Triton-X100 (Sigma X100-500ML), 2mM bis(sulfosuccinimidyl)suberate (BS3), 10mM DTT) for 5 minutes. Additional 4ml of PBS-BS3 (PBS, 2mM BS3, 10mM DTT) was then added, and cells were incubated on ice for 15 minutes. Cells were pelleted at 500xg, 4 °C for 5 minutes and resuspended in 500ul nuclease-free water supplemented with 0.1% SUPERase In and 10mM DTT. 3ml of 0.05N HCl was added for further permeabilization. After 3 minutes of incubation on ice, 3.5ml Tris-HCl, pH 8.0, and 35ul of 10% Triton X-100 were added to each tube to neutralize the HCl. After spinning down at 4 °C, 500xg for 5 minutes, cells were finally resuspended in 400ul PSB-DTT at the concentration of ~2e6 cells/100ul (PBS, 1% SUPERase In, 1% Bovine Serum Albumin (BSA), 1mM DTT), mixed with 10% DMSO, and were slow-frozen and stored in -80 °C. Fixed cells were thawed, then mRNA and sgRNA within each individual cell were indexed by indexing reverse transcription, indexing ligation, and finally indexing PCR. SgRNA libraries were further separated and enriched by another round of PCR. A step-by-step experimental protocol of PerturbSci-Kinetics library preparation is included in the supplementary material of the publication.