GSM2616861: DFL_E12_del2_Hoxd13VP; Mus musculus; OTHER
Micro-dissected proximal and distal segments of E12.5 limbs either from wild type or 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, Isl_II forward primer: GCATTCATCAAGCTGTGATTAGCA, Isl_II reverse primer: TCCCATAATATGTAGACTGTAGTGTTGC. For sample 9 we used a series of four different barcoded primers for Hoxd13 forward such as barcode 1: AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTATCGAAAAATCCTAGACCTGGTCATG; iHoxd13 forward:  barcode 2: AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTGACAAAAAATCCTAGACCTGGTCATG; iHoxd13 forward: barcode3: AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTTGACAAAAATCCTAGACCTGGTCATG; and iHoxd13 forward: barcode4: AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCTGGTAAAAAATCCTAGACCTGGTCATG
GEO Accession
GSM2616861