Instrument: Illumina Genome Analyzer IIx
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: Preparation of 4C samples was performed as described (van de Werken, 2012) with some modifications. Freshly collected tissues were incubated with collagenase (0.0065% collagenase I in 10% FBS/PBS for cortex, and 1.33 mg/ml collagenase P in HBSS for pancreas) for 40 min at 37ºC in a shaker. The resulting cell suspension was filtered through a 40-μm cell strainer in order to get a single cell preparation. Cells were centrifuged at 1,100 rpm for 5 min at RT and the cell pellet was resuspended in 10 ml of fixing solution containing 2% formaldehyde in 10% FBS/PBS. After 10 min, 0.125 M glycine was added to stop fixation and the mixture was incubated for 5 min at RT. Cells were pelleted and nuclei isolated upon incubation in cold lysis buffer in the following conditions: For the brain, 10 min in 50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% TX-100 + protease inhibitors cocktail; for the pancreas, 40 min in 50 mM Tris pH 7.5, 50 mM NaCl, 5 mM EDTA, 1% NP40, 2% TX-100 + protease inhibitors cocktail. Proper lysis was determined by methyl green-pyronin staining. 4C-seq libraries were amplified using long primers with 18-21 bp homology to the bait sequence and Illumina paired-end adapter flanks. HindIII and CvIQ (NEB) were used as as first and second cutters, respectively for preparation of the 6-cutter libraries. DpnII and CvIQ (NEB) were used as first and second cutters, respectively for preparation of the 4-cutter libraries. Pairs of samples were sequenced together thus the raw and processed data files are shared among samples. The 4cseqpipe software identifies the viewpoints and the possible cut points out of the raw data files and flags them in the processed files.