title: GSM2092710: PLB_E12_Hoxa13het-Hoxd13KO_Hoxd11VP; Mus musculus; OTHER
library_construction_protocol: Micro-dissected proximal and distal segments of E12.5 limbs either from wild type, Hoxa13het- Hoxd13KO or Hoxa13d13-dKO mutant embryos were dissected, dissociated with collagenase (Sigma Aldrich/Fluka) and filtered through a 35 micron mesh to isolate single cells. Cells were fixed with 2% formaldehyde for 10 min at room temperature and the reaction was quenched on ice with glycine. Cells were further lysed with 10 mM Tris pH 7.5, 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 1x Protease inhibitor cocktail to isolate nuclei and stored at -80°C. Nuclei from pools of at least 8 distal or proximal limbs were digested with NlaIII (New England Biolabs) and ligated with T4 DNA ligase HC (Promega) in diluted conditions to promote intramolecular ligation. Sequences ligated to fragments of interest were digested again with DpnII (New England Biolabs), ligated with T4 DNA ligase HC (Promega) in diluted conditions. These templates were amplified using Expand long template (Roche) and inversed PCR primers flanked with adaptors allowing multiplexing.  Hoxd13_inverse_forward:AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTAAAATCCTAGACCTGGTCAT Hoxd13_inverse_reverse:CAAGCAGAAGACGGCATACGAGGCCGATGGTGCTGTATAG Hoxd11_inverse_forward:AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTAGCATACTTCCTCAGAAGAGGC Hoxd11_inverse_reverse:CAAGCAGAAGACGGCATACGACTAGGAAAATTCCTAATTTCAG