SisterC: A novel 3C-technique to detect chromatin interactions between and along sister chromatids

Marlies E. Oomen Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA Adam K. Hedger RNA Therapeutics Institute and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA. Jonathan K. Watts RNA Therapeutics Institute and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA. Job Dekker (  job.dekker@umassmed.edu ) Program in Systems Biology, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA, USA. Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.

Phase lock gel, light 2 ml (5 Prime #2302820) DNA LoBind tubes, 1.5 ml (Eppendorf #022431021) Amicon® Ultra -0.5ml 30K (Millipore #UFC5030BK) ThermoMixer C (Eppendorf #2231000667) Procedure 1. Synchronization Note: We describe here the protocol to perform SisterC in budding yeast. As wild type yeast cannot incorporate BrdU, we recommend to use a mutant strain, e.g. YLV11 strain, which expresses the nucleoside transporter hENT and nucleoside kinase DmdNK 5 . Although not tested, this protocol can be modi ed for use in mammalian cell lines and other model systems. The synchronization protocol is listed here is speci c for the YLV11 strain. Cells are grown in YP media with 2% galactose and 100µM Thymidine at 30°C unless stated otherwise. Note: Cell cycle state and BrdU incorporation can be assessed qualitatively by ow cytometry of all timepoints during synchronization using anti-BrdU antibody staining. Quantitative measurement of BrdU incorporation e ciency can by done by HPLC detection of genomic DNA isolated from cultures.
2. Crosslinking of cells extraction without ethanol precipitation. Add 1μl of RNAseA (1 mg/ml) to the aqueous phase and incubate for 15 min at 37°C. Check quality of the sample by running it on 0.8% agarose gel. DNA is expected to run as a single high molecular weight band. 8. Add 3.8 μl of 1% SDS to each Hi-C tube (~380 µl total). Mix carefully by pipetting up and down (0.01% SDS nal). 9. Incubate at 65°C for 5 minutes exactly to open chromatin, place tubes on ice immediately after 10. Add 43 μl of 10% Triton X-100 to the Hi-C-tube (423 µl total) to quench the SDS (1% Triton nal). Mix gently by pipetting up and down Avoid making bubbles.
NB! The addition of the following ingredients depends on the enzyme and its concentration. N.B! In the nal mix we have DNA, the reagents used in this step, and free nucleotides including the biotin-14-dATP. In order to clean DNA for sonication we wash the DNA using amicon column. In this step a lot of DNA can be lost, so when you have a low input library consider skipping this step and risk a low e cient sonication.

A-tailing
Note: After performing the A-tailing reaction, make sure to continue immediately with step 13 (illumine adapter ligation) Note: If chromatin digestion in step 3 was performed with an alternative restriction enzyme, the enzyme used at this step needs to be changed accordingly as well.
5. Run the pcr titration and test digest on a 2% agarose gel.
Note: Choose an optimal number of cycles and number of PCR reactions for the nal ampli cation of the library for deep sequencing. The cycle number should be chosen so that the PCR ampli cation is in the linear range and the expected size distribution is preserved (over cycling will shift the size distribution of the library towards higher molecular weight products). The number of PCR reactions should be calculated