ChIP-seq: D4 EBs (1-day induced and control Pax3 mouse cells) were trypsin-treated at 37°C for 1 minute with gentle shacking and reaction was inhibited by adding 10%FBS/PBS. 1-day induced Pax3 NIH3T3 cells and 6-day induced Pax3 mouse cells growing in monolayer were harvested by incubation with trypsin for 1-2 minutes. Single cells were washed once with PBS, resuspended in 10%FBS/PBS and supplemented with formaldehyde (final concentration 1%) for crosslinking of protein-DNA complexes (10 minutes at RT) followed by quenching with glycine and staining with PDGFRα-PE antibody. PDGFRα+ cells were sorted using FACSAriaII, snap-frozen in liquid nitrogen and stored at -80°C if not processed immediately. Cell pellets were incubated in lysis buffer LB1 supplemented with protease inhibitors (50mM HEPES KOH pH7.5, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X-100 + Complete-mini - Roche) for 10 minutes at +4°C followed by incubation in buffer LB2 supplemented with protease inhibitors (10mM TRIS HCl pH 8, 200mM NaCl, 1mM EDTA, 0.5mM EGTA + Complete-mini - Roche) for 10 minutes at +4°C. Cell pellet was then resuspended in LB3 supplemented with protease inhibitors (10mM TRIS HCl pH 8, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Sodium Deoxycholate, 0.5% N-lauroylsarcosine + Complete-mini - Roche) and then sonicated. Cells were sonicated with a Branson sonicator at 18% power for 1 minute with intervals of 10 sec ON-10 sec OFF to achieve an average chromatin size of 300bp. After shearing, samples were centrifuged for 10 minutes at 16000g and snap frozen in liquid nitrogen if not processed immediately. 10-15µg (histone mark) or 25-40µg (Ldb1, Smc1, Ctcf) of chromatin were diluted to 250µl and 500µl, respectivley, and then precleared for 4h at 4°C with 20µl of BSA-blocked Protein A (or Protein G)-conjugated sepharose beads (GE healthcare). Samples were supplemented with 1/10 volume of 10% Triton X-100 and incubated overnight with the indicated antibody. Immune complexes were recovered by incubation with 20µl of BSA-blocked Protein G-conjugated sepharose beads for 4h at 4°C and then washed 5 times with RIPA wash buffer (50mM HEPES KOH pH7.5, 500mM LiCl, 1mM EDTA, 1% NP40, 0.25% Triton X-100, 0.7% Sodium Deoxycholate) and one time with TEN buffer (10mM TRIS HCl pH 8, 1mM EDTA, 50mM NaCl). Immunoprecipitated chromatin was recovered by incubating beads with 200µl of Elution buffer (50mM TRIS HCl pH 8, 10mM EDTA, 1% Sodium Dodecyl Sulfate) for 20 minutes at 65°C. Chromatin from IP and Input (equivalent to 1% of starting material) was reverse crosslinked overnight at 65°C, then diluted 1:1 with TE (10mM TRIS HCl pH 8, 1mM EDTA) supplemented with 4µl of RNaseA 20mg/ml and incubated for 2 hours at 37°C followed by Proteinase K treatment (4µl of 20mg/ml stock for each sample) for 30 minutes at 55°C. DNA was purified by Phenol-chloroform-isoamyl alcol extraction (twice) followed by chloroform extraction, then supplemented with 1/10 of volume of 3M Sodium Acetate pH 5 and 1.5µl of Glycogen and precipitated with 2 volumes of 100% Ethanol at -80°C for >1 hour. Followed 30 minutes centrifuge at 16000g, pellet were washed with 75% ethanol, air dried and dissolved in 45µl H2O. ChIP-seq libraries were generated following a gel-free protocol using AMPure XP beads (Beckman Coulter) for all the purification and size selection steps. 10ng or less of DNA were end repaired using End-it DNA end repair (Epicentre), then A-tailed using Klenow Fragment (3'→5' exo- NEB) followed by adapter-barcode ligation using T4 DNA ligase (Enzymatics). Illumina compatible adapter-barcodes were purchased from BIOO scientific. After ligation, DNAs were negatively size selected using 0.5x Ampure XP beads and unbound DNAs were positively size selected by adding 0.4x Ampure XP beads (this step allows for retention of DNA fragments ranging 200-500bp). Libraries were amplified using Phusion High Fidelity PCR master mix 2x (NEB) with a 16 cycles program. Libraries from Ldb1, histone marks in 6-day induced cells and H3K4me1 in dTAD and dTAD-Ldb1 immunoprecipitations were generated using the NEBNext DNA library prep kit (NEB). HiChIP libraries. Long range interactions involving Pax3 binding sites were analyzed following the detailed protocol described by the Chang group. Briefly, non-induced, 1-day and 6-day Pax3-induced cells (respectively mesoderm, paraxial mesoderm precursors and myogenic progenitors) and 6-day+shSCR and 6-day+shLdb1 cells were harvested using trypsin for 1-2 min followed by inactivation with PBS/10%FBS, centrifuged 5 min at 400g, washed with PBS twice and then counted. 13 million cells, diluted to 1million/ml in PBS, were crosslinked for 15 min using methanol-free formaldehyde (Pierce – Thermoscientific). In situ contact generation was performed by digesting chromatin with MboI (NEB) followed by blunting with dNTP mix containing biotin-dATP (Thermo Fisher, 19524016) and ligation using T4 DNA ligase (NEB). After centrifugation, nuclei were resuspended in 880μL in Nuclear Lysis Buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS, Complete Protease inhibitor - Roche), transferred in a Millitube and sonicated using a Covaris S220 (parameters: fill level = 10, duty cycle = 5, PIP = 140, cycles/burst = 200, time = 4 min). Chromatin was cleared by centrifugation, diluted 1:2 in ChIP Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 7.5, 167 mM NaCl), precleared for 4h at +4°C with 75µl of Protein G-conjugated magnetic beads and then incubated with 30µg of anti-Pax3 and anti-Ldb1 antibodies overnight at +4°C. Immune complexes were recovered by incubation with 60µl of BSA-blocked Protein G-conjugated sepharose beads for 5h at +4°C, then washed 5 times with RIPA wash buffer (50mM HEPES KOH pH7.5, 500mM LiCl, 1mM EDTA, 1% NP40, 0.25% Triton X-100, 0.7% Sodium Deoxycholate) and one time with TEN buffer (10mM TRIS HCl pH 8, 1mM EDTA, 50mM NaCl). Due to the sample volume during the Pax3 IP, Protein-G:Pax3-chromatin bound beads were splitted in two 1.5ml tubes during the previous procedure. Immunoprecipitated chromatin was recovered by incubating beads with 200µl/tube of Elution buffer (50mM TRIS HCl pH 8, 10mM EDTA, 1% Sodium Dodecyl Sulfate) for 30 minutes at 65°C. ChIP samples and Input (equivalent to 1% of starting material) were reverse crosslinked overnight at 65°C, then diluted 1:1 with TE (10mM TRIS HCl pH 8, 1mM EDTA) supplemented with 4µl of RNaseA 20mg/ml and incubated for 2 hours at 37°C followed by Proteinase K treatment (4µl of 20mg/ml stock for each sample) for 30 minutes at 55°C. DNA was purified by Phenol-chloroform-isoamyl alcol extraction (twice) followed by chloroform extraction, then supplemented with 1/10 of volume of 3M Sodium Acetate pH 5 and 1.5µl of Glycogen and precipitated with 2 volumes of 100% Ethanol at -80°C for >1 hour. Following 30 minutes centrifuge at 16000g, pellets were washed with 75% ethanol, air dried and the combined DNA pellet were dissolved in 40µl H2O. Before proceeding with the biotin capture and transposase mediated library generation, ChIP samples were analyzed by qPCR to ensure enrichment at Pax3 sites vs control region. Samples were quantified using Pico-green (Invitrogen), resuspended in binding buffer and then incubated with 20µl of Streptavidin T1-conjugated magnetic beads (Invitrogen). After washing, DNA-bound streptavidin beads were incubated with 1.5µl of Tn5 transposase in a final volume of 50µl for 10 minutes at 55°C. This amount of transposase was selected based on the picogreen results of the ChIP, which yielded 25ng of DNA. Following quenching and washing, transposed DNA was incubated 5 minutes at 65°C with activated NEBNext UltraII QS Taq (+Illumina adaptor/barcodes) to allow primer extension and then amplified for 5 cycles (15 seconds 98°C followed by 1.5 minutes at 65°C). Additional cycles for library amplification were determined by qPCR using T5-T7 primer