library_construction_protocol: Automated CUT&Tag sequencing To assess multiple histone marks and genome protein occupancies in each NSPCs preparation from an individual embryo, we followed an automated CUT&Tag protocol developed by Henikoff and colleagues (Janssens et al., 2021), which allows parallel processing of many samples detecting many targets in a 96-well plate format. We used a liquid handling robot (Bluewasher) for removing liquids and adding wash buffers to plates. For these CUT&Tag experiment, 1x106 NSPCs were cultured in a 20 cm dish for 3 days under proliferating conditions or 2x106 NSPCs were plated onto 10 cm dishes and cultured for 3 days under differentiation conditions. Both proliferating and differentiating NSPCs underwent chemical dissociation with Accutase (STEMCELL Technologies, 07920). NSPCs were collected, counted and washed. Cells were bound to concanavalin-A beads and permeabilised using 0.05% digitonin and 0.01% NP-40A. Permeabilised cells were counted, and 50,000 cells were aliquoted for each CUT&Tag reaction in a 96 well plate well. Cells were stained with primary antibody at 1:50 dilution overnight. Cells were stained with nanobody-Tn5 at 1:50 and tagmentation reactions were performed for 1 hour at 37˚C. Tagmented fragments were isolated using SPRI beads. Optimal indexing PCR cycle number was determined by qPCR and all reactions for the same primary antibody were indexed using dual indexing primer (Mezger et al., 2018) for 9-20 PCR cycles. Sequencing libraries concentration ranged between 0.5-15 ng/µl as determined by QuantiFluor dsDNA. Indexed sequencing libraries were pooled and sequenced on NextSeq2000 using one P3 (1.2B reads) and three P2 (400M reads) kits producing 50 bp paired end reads and 8 bp dual indexing reads and standard Illumina sequencing primer. Some samples were re-amplified using the same number of indexing PCR cycles to acquire more material for sequencing. The antibodies to the following antigens were used: H3K9ac (Cell Signaling Technology, 9649), H3K14ac (Abcam, ab52946), H3K23ac (Millipore, 07355), MLL1 (Cell Signaling Technology, 14689), H3K4me3 (Active Motif, 36090), RNA polymerase II subunit A phosphorylated on Ser2 [POLR2A; also known as Phospho-Rpb1 CTD; (Cell Signaling Technology, 13499)], IgG (Epicypher, 13-0042).