GSM8534989: 4Cseq-1h30-MLN-rDNA; Homo sapiens; OTHER
10-15 million cells were collected, washed once with 1X PBS, and cross-linked using freshly prepared 2% formaldehyde solution by tumbling for 10 min at room temperature. The fixation was stopped by adding glycine and immediately transferring the tubes to ice. The cross-linked cells were collected at 300g for 5 min at 4℃ and washed once with 1X PBS. After removing the supernatant, the pellet was snap-freeze in liquid nitrogen and stored at -80°C. Chromatin fixation, cell lysis and 4C-seq procedure were done as described in Miranda et al, Methods in Molecular Biology 2022 using 10-15 million cells per cell experiment. MboI (New England Biolabs) was used as the primary restriction enzyme and NlaIII (New England Biolabs) as the secondary restriction enzyme. 4C-seq sequencing libraries using the rDNA viewpoint, which is present in the human genome in several hundred copies, were generated using a 2-step amplification approach with reduced amounts of input, as described in Haarhuis et al, Cell 2017 and Krijger et al, Methods 2020. In the first step, a total of 120ng of 4C material was amplified using the Expand Long Template PCR System (Roche Diagnostics) in 12 reactions in parallel for 12 cycles. All reactions were pooled, followed by clean-up of 1/6th of the material using Agencourt Ampure XP Beads (Beckman Coulter) to remove fragments smaller than 200 bp. In the second step, 20% of the purified PCR products were further amplified in 2 reactions using the Expand Long Template PCR System and universal index adapters for 20 cycles. Amplified 4C-seq libraries were again purified with Agencourt Ampure XP Beads to remove fragments smaller than 200 bp. Quality and size distribution of the PCR libraries was verified by Qubit ds DNA BR kit (Thermo Fisher Scientific) and Tapestation Genomic DNA reagents (Agilent).