Laminin

Using optimal conditions for dissociation of the epidermal cells (incubation in Dispase I in M199 (0.6–1.0 U/ml) for 10 min), ostracod epidermal cells were plated on different substrates (i.e. cells were pooled from 30 ostracods per substrate-coated well) to promote adherence during culture in the optimised M199 medium described above. Substrates included collagen type I (48-well; Sigma), collagen type IV (48-well; Sigma), laminin (24-well; Corning), fibronectin (24-well, BioCoat; Corning), Matrigel (48-well; Sigma) and chitosan. For chitosan coatings, 4% (w/v) chitosan (>90% degree deacetylation; Sigma) in 2% (v/v) acetic acid was autoclaved, applied to 48-well plates and allowed to dry for 24 h to form a thin film. The acidity of the films was neutralised with 0.5 M NaOH and washed repeatedly with Hank’s balanced salt solution until film pH returned to a physiological range (pH = 7.4).

Trypsin-EDTA (Cat# 25-053-CI), PBS (Cat# 21-031-CV), and laminin (Cat# 354232) were purchased from Corning. Recombinant EGF was purchased from Life Technologies (Cat# PHG0311). IGF was purchased from Sigma-Aldrich (Cat# I3769). PIM447 was purchased from Selleck Chemicals (Cat# S7985). AZD1208 was acquired from AdooQ Bioscience (Cat# A13203-750). DMSO was purchased from Thermo Fisher Scientific (Cat# 97064-724). Cycloheximide was purchased from VWR (Cat# 97064-724), and doxycycline was purchased from Sigma-Aldrich (Cat# D9891-5G). Recombinant myelin basic protein (MBP) was a gift from Dr. Greg Rogers, and recombinant ABI2 protein was purchased from Origene (Cat# TP300637). Radio-labeled ATP was purchased from Perkin Elmer (Cat# BLU502A). The antibody to ABI2 was purchased from Bethyl Laboratories (Cat# A302-499A-M). GFP (Cat# 2956S), HA (Cat# 3724S), HIF-1a (Cat# 14179S), p-IRS1 [S1101] (Cat# 2385S), PIM1 (Cat# 3247S), and WAVE2 (Cat# 3659S) antibodies were purchased from Cell Signaling Technology. The antibody for actin was purchased from BD Biosciences (Cat# 61656). PIM1 (Cat# ab75776) and Ki67 (Cat#ab833) antibodies used for immunohistochemistry were purchased from Abcam.

The V6.5 mESCs and iPSCs were cultured on irradiated primary mouse embryonic fibroblasts (MEFs) under standard serum/leukemia inhibitory factor (LIF) conditions (KO DMEM containing 15% fetal bovine serum (FBS), supplemented with 1× penicillin/streptomycin, 1× GlutaMAX supplement, 1× nonessential amino acids, 0.05 mM β-mercaptoethanol (all from Gibco) and 1,000 U ml^–1 LIF).
For ChIP–seq, TT-SLAM–seq and RNA-seq experiments, mESCs were depleted from MEFs by incubating them on gelatin-coated cell culture plates for 45 min at 37 °C, allowing MEFs to attach while mESCs remain in suspension. MEF depletion was performed twice, after which mESCs were seeded on gelatin-coated plates and maintained in serum/LIF conditions with 2,000 U ml^–1 LIF.
For RNA-FISH combined with IF, MEF-depleted cells were grown on round 18-mm glass coverslips (Roth LH23.1). Coverslips were coated with 5 µg ml^–1 of poly-l-ornithine (Sigma-Aldrich, catalog no. P4957) for 30 min at 37 °C and with 5 µg ml^–1 of Laminin (Corning, catalog no. 354232) overnight at 37 °C.
To perturb RNAPII condensates, cells were treated for 30 min with 1.5% 1-6 HD (Sigma) in serum/LIF conditions with 2,000U ml^–1 LIF (Extended Data Fig. 3c,d).

Primary cranial or trunk NC cells were seeded at 10,000 cells/cm² onto tissue culture-treated glass slides coated with 50 µg/ml poly-D-lysine (Sigma-Aldrich) and 10 µg/ml laminin (Corning). Cells were grown in neuronal differentiation medium: Neurobasal-A Medium (Gibco), 2 mM GlutaMAX (Gibco), SM1 neuronal supplement (STEMCELL Technologies), 100 U/ml penicillin and 100 µg/ml streptomycin (Corning). The medium was sterile filtered, then supplemented with 100 ng/ml mouse nerve growth factor 2.5S (NGF; MilliporeSigma) and 50 ng/ml recombinant human neurotrophin-3 (NT3; MilliporeSigma). Half the medium was exchanged every other day.