4C sample preparation was performed as previously described by Splinter et al. using NlaIII (NEB) and Csp6I (Thermo)34 (link). Viewpoint primers were selected from a previously reported database35 (link).
Primers sequences were concatenated with Illumina compatible adapter sequences and unique barcodes and synthesized as Ultramer oligos (IDT). Final library amplification was set up on ice using 3.2 µg of purified 4C sample DNA, 2.5 µM primers, and Ultra II Q5 Master Mix (NEB) split into 16 × 50 µl PCR reactions with extension at 65 °C. Amplification products were purified using 1.5× Ampure XP (Beckman) bead purification. Size distributions were established using a Bioanalyzer DNA 12000 Assay (Agilent).
Paired-end sequencing reads were generated as above and analyzed with the 4C-ker pipeline as previously described to identify genomic loci with differential chromosome contacts between conditions36 (link).
Primers sequences were concatenated with Illumina compatible adapter sequences and unique barcodes and synthesized as Ultramer oligos (IDT). Final library amplification was set up on ice using 3.2 µg of purified 4C sample DNA, 2.5 µM primers, and Ultra II Q5 Master Mix (NEB) split into 16 × 50 µl PCR reactions with extension at 65 °C. Amplification products were purified using 1.5× Ampure XP (Beckman) bead purification. Size distributions were established using a Bioanalyzer DNA 12000 Assay (Agilent).
Paired-end sequencing reads were generated as above and analyzed with the 4C-ker pipeline as previously described to identify genomic loci with differential chromosome contacts between conditions36 (link).
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