GSM3640292: lacZ_inv1112d13Z_G44 (4C-seq); Mus musculus; OTHER
Metanephros were micro-dissected and dissociated from homozygous E13.5 mutant embryos (HoxD d13lacZ and HoxD inv(11-12)d13lacZ). Metanephric kidneys were dissociated with collagenase (Sigma Aldrich/Fluka). Cells were fixed with 2% formaldehyde (in PBS/10%FBS) 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 EDTA, 1x Protease inhibitor cocktail to isolate nuclei and stored at -80°C. Nuclei from pools of at least 10 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. Samples were digested again with DpnII (New England Biolabs) and 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 barcodes allowing multiplexing. The Illumina adaptors used were 5'-AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' for the inverse-forward primers located at the NlaII site and 5'-CAAGCAGAAGACGGCATACGA-3' for the inverse-reverse primers located at the DpnII site. Barcodes (4bp) were added between the illumina adaptor and the specific NlaIII primers when the same viewpoints were multiplexed in the same run. The barcode is mentioned in the raw file name being: C14 (CAGA), G34 (GATC), C44 (CTGT) and G44 (GCCA). Specific primer information for each viewpoint is displayed in the related publication (Darbellay et al, 2019).
GEO Accession
GSM3640292