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Figure 1 | BMC Genomics

Figure 1

From: Identifying multi-locus chromatin contacts in human cells using tethered multiple 3C

Figure 1

Overview of TM3C experimental protocol and mapping of paired-end reads to human genome. 1. Cells are treated with formaldehyde, covalently crosslinking proteins to one another and to the DNA. The DNA is then digested with either a single 4-cutter enzyme (DpnII) or a cocktail of enzymes (AluI, DpnII, MspI, and NlaIII). 2. Melted low-melting agarose solution is added to the digested nuclei to tether the DNA to agarose beads. Thin strings of the hot nuclei plus agarose solution is then transferred to an ice-cold ligation cocktail overnight. 3. After reversal of formaldehyde crosslinks and purification via gel extraction, the TM3C molecules are sonicated and size-selected for 250 bp fragments. 4. Size-selected fragments are paired-end sequenced (100 bp per end) after addition of sequencing adaptors. 5. Each end of paired-end reads are mapped to human reference genome. If both ends are mapped then the pair is considered a double and retained because it is informative for genome architecture. 6. Read ends that do not map to the reference genome are identified and segregated according to the number of cleavage sites they contain for the restriction enzyme(s) used for digestion. 7. Reads with exactly one cleavage site are considered for the second phase of mapping. These reads are split into two from the cleavage site and each of these two pieces are mapped back to the reference genome. 8. Read pairs with either one or both ends not mapped in the first mapping phase are reconsidered after second phase. Depending on how many pieces stemming from the original reads are mapped in the second phase, such pairs lead to either no informative contacts, doubles, triples or quadruples.

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