GSM4906107: Nell2_E12_DFL; Mus musculus; OTHER
Cells were 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 DpnII (New England Biolabs) and ligated with T4 DNA ligase HC (Promega) in diluted conditions to promote intramolecular ligation. Samples were digested again with NlaIII (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 adaptors allowing multiplexing (Table III). Barcodes (4bp) were added between the Illumina adaptor and the specific DpnII primers. Libraries were prepared by means of 8–10 independent PCR reactions using 70–100 ng of DNA per reaction. PCR products were pooled and purified using the PCR purification kit (Qiagen).  The illumina adaptors used were 5'-AATGATACGGCGACCACCGAACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' for the inverse-forward primers located at the DpnII 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 DpnII primers when the same viewpoints were multiplexed in the same run. Specific primer information for each viewpoint is displayed in the related publication (Beccari L et al, 2021). Undigested and self-ligated (and for Ano6 identified off-targets) fragments were discarded according to the sequences provided as an associated file named "4C-seq_filtered_out_sequences.txt". Multiplexed libraries were sequenced on Illumina HiSeq 2500 to obtain 100 bp single-end reads.