Increasing the throughput of PoreC reveals multiway chromatin contacts that reflect regional topologies of single chromosomes. NBThighlight
Fifteen million GM12878 or K562 cells were spun down and resuspended in 10 ml of fresh medium. Cells were fixed by adding 278 μL of 37% formaldehyde and incubated for 10 minutes at room temperature . The reaction was stopped by adding 894 μL of 2.5 M glycines. The cell suspension was incubated for five minutes at RT, followed by 10 minutes on ice. Fixed cells were pelleted by centrifugation at 1000 ×for 5 minutes at 4 °C and then gently washed twice with 5 ml of ice-cold 1× PBS.
For version 1 HiPore-C, we repeated digestion by adding 20 µL of 10% SDS and 10 µL of 20 mg/ml proteinase K to 170 μL of DNA solution. The mixture was incubated at 63 °C for 1 hour to digest the remaining associated protein and purified as in the first round. Proteinase digestion and reverse crosslinking can be repeated for another round. The final library DNA was dissolved in 30 μL of Buffer EB.
For version 2 HiPore-C, we digested samples for an additional round with pronase and then purified library DNA as described in the Pore-C and HiPore-C version 1 protocols. The final library DNA was dissolved in 30 μL of Buffer EB.3-4 ug of purified DNA per sample was used as input material for ONT sequencing library preparation. DNA was size selected using the PippinHT system .
. on GM12878 and K562 cell lines and sequenced these libraries on the PromethION platform for comparison with the HiPore-C.Nanopore sequencing raw signals were converted to DNA sequences using the high-accuracy model “dna_r9.4.1_450bps_hac_prom.cfg” of Guppy v4.5.3 software and reads with quality scores less than 7 were discarded. Sequencing statistical analysis was conducted using NanoPlot
. 5mC methylation sites were called using Megalodon v2.3.4 with the ‘–guppy-config res_dna_r941_prom_modbases_5mC_v001.cfg –outputs mod_basecalls –mod-motif m CG 0 –devices cuda:0 –processes 48 –overwrite’.
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