Targeted Perturb-seq of regulators of the transcriptional network downstream of RAF-MAPK signaling [perturb_seq]

In this study we use a (targeted) perturb seq experiment in which we knock out 22 transcription factors downstream of MAPK signalling in order to reconstruct the transcriptional network downstream of RAF/MAPK signaling. Overall design: HEK-RAF-ER were transduced with a pooled CRISPR/Cas9 library targeting 22 transcription factors and the RAF-ER transgene with 4 guides each. Additionally, 10 non-targeting controls and 10 safe-cutters were included as negative controls. After a period of editing, the RAF/MAPK pathway was induced by 4OHT (tamoxifen), and single cell transcriptome gene expression and CRISPR libraries were generated using the 10x Chromium Single Cell 3' V3 with Feature Barcoding technology. Additionally, we performed targeted PCR amplification of 140 genes that yielded better coverage of these genes. These dial-out libraries were sequenced seperately.