Generating GFP-TCOF1 and GFP-DDX21 Constructs

For generating GFP–TCOF1 and GFP–DDX21 expression vectors, gBlocks gene fragment (IDT) encoding the human TCOF1 and DDX21 coding region were synthetized and cloned into a GFP-expressing and tetracycline-inducible PiggyBac vector (SBI). The PiggyBac was stably integrated into pluripotent cells and several clones were expanded for further analyses. For generation of the cNCCs, we used our previously published protocol^{28 (link),29 (link)} (described below). To express the transgenes, doxycycline was added to the medium at a concentration of 2.5 μg ml⁻¹ for 24–48 h. For the generation of Tcof1 mutant ES cells and mouse ES cells, single guide RNAs (sgRNAs) were developed at http://crispr.mit.edu/ and cloned into the PX458 vector (Addgene, 48138). Cells were transfected with Lipofectamine 2000 (Life Technologies), and sorted on the basis of GFP The resulting clones were expanded and screened by PCR to search for Tcof1 loss-of-function alleles. I-Ppol construct (Addgene, 32565) was developed in M. Kastan’s laboratory. AsiSI was synthetized as a gBlock gene fragment (IDT).