GSM8530773: Forebrain_Hoxa13_4C-seq_rep1; Mus musculus; Hi-C
For each sample, 2 million cells were cross-linked in 1% formaldehyde for 10 min at room temperature and quenched with glycine to a final concentration of 0.2 M. Cross-linked samples were rinsed once with PBS. Cells were lysed using a buffer containing 10 mM Tris-HCl (pH 7.5), 10 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA and Protease Inhibitor Cocktail. NlaIII (cat. #R0125, NEB) was used as primary restriction enzyme, DpnII (cat. #R0543) was used as secondary restriction enzyme. 4C-seq libraries were prepared by sequential NlaIII and DpnII digestion and ligation of T4 DNA ligase covering the mouse HoxA clusters. For each viewpoint, a total of 1 ug of each purified 4C-seq template was amplified using 16 individual PCR reactions with inverse primers including Illumina Universal adaptor sequences. The sequencing libraries were qualified with Qubit 2.0 and Agilent 2100, and sequenced on an Illumina HiSeq platform for each library.