Multiplexing genome architecture mapping reveals orthogonal insights to HiC. NBThighlight
. Cryosections of 220 nm were cut with glass knives using a Leica FC7 ultracut cryotome, collected in sucrose droplets and transferred to steel frame PEN membrane slides for ultraviolet treatment for 45 min prior to use. Slides were washed in sterile-filtered 1× PBS , then with sterile-filtered water . Cresyl violet staining was performed with sterile-filtered cresyl violet for 10 min, followed by two washes with water and air dried for 15 min.
Whole genome amplification of DNA from microdissected nuclear profiles was performed with the Sigma WGA4 kit using a liquid handling robot . We note that several consecutive Sigma WGA4 kits stopped working in 2017 for GAM data production, and we currently recommend a more affordable in-house whole genome amplification protocol
. A total of 14.5 μl lysis and fragmentation master mix was added to each well of a 96-well plate, caps with microdissected material were used to close the wells and then the plate was inverted and centrifuged upside down at 3,000 ×for 2 min such that the fragmentation master mix was collected in the cap. Plates were incubated upside down for 4 h at 50 °C then inverted and centrifuged the right way up at 3,000 ×for 2 min to collect the extracted DNA in the bottom of the well.
Amplified DNA was purified using Ampure XP beads . The beads were mixed with 77 μl amplified sample in a fresh 96-well plate and incubated at room temperature for 5 min. The plate was placed on a magnetic stand for 5 min; then the supernatant was discarded and the beads were washed twice with 200 μl freshly prepared 80% ethanol. After the second ethanol wash was discarded, the beads were air dried for 5 min and then resuspended in 45 μl HO and incubated at room temperature for 5 min.
Libraries were prepared using the Illumina Nextera library preparation kit following the manufacturer’s instructions. The DNA concentration of the final libraries was determined using a Picogreen fluorescence assay , and libraries were pooled at equimolar concentration, ready for sequencing on an Illumina NextSeq machine.Multiplex-GAM sequencing reads were aligned to the mouse mm9 genome assembly using Bowtie2 v2.1.0, and PCR duplicates were filtered using Samtools v0.9.0.
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