Maheshri:3C

Adapted from Singh, Ansari and Hampsey, "Detection of gene loops by 3C in yeast" Methods (2009) 48:361-367.

Day 1 Grow cells 20ml ON in YPD

Day 2 Harvest cells 1. Inoculate 50ml culture of YPD starting from OD 0.1 (from 20ml culture) 2. Grow to OD 0.8 3. Add 1.5ml formaldehyde (1% concentration) 4. Incubate 15min room temp with shaking

5. Add 2.5ml 2.5M glycine 6. Incubate 5min room temp with shaking

7. Centrifuge 3000g 4C for 5min 8. Wash cells with ice-cold 10ml of TBS+1% Triton X-100. Spin 3000g at 4C for 5min and remove supernatant. 8. Repeat 7-8 twice.

9. Optional: Freeze pellet -80C (up to 3 days)

Lyse cell and digest chromatin

1. Optional (if frozen) a. Thaw cells on ice b. Spin down cells and remove supernatant

2. Resuspend pellet in 500μL FA lysis buffer and transfer to 2ml screw cap tude (flat bottom tubes preferred over eppi tubes) 3. Add 5μL 100mM PMSF (kept in -20C)

4. Add 400-500μL glass beads 5. Vortex cells 40min at 4C at full speed Tubes need to be taped to the vortex machine to prevent falling

6. Prepare 5ml syringe-15ml tubes to collect lysates 7. After vortexing, punch hole at the bottom of screw cap tube and cap with 25G needle 8. Centrifuge the tube in syringe 1000rpm 3min 4C 9. Transfer lysate to 1.5ml eppi tube

10. Spin down 4C at max speed 15min 11. Discard supernatant and resuspend with chromatin pellet with 500μL FA lysis buffer + 5μL 100mM PMSF 12. Spin down 10min 13000g 4C and remove supernatant

13. Resuspend pellet in 800μL 10mM Tris HCL (pH7.5) 14. Aliquot 80 μL into one tube Making small aliquot seems to prevent excessive clumping

Enzyme digestion

1. Prepare digest - 80 μL chromatin - 10 μL Alu1 (4 cutter) - 10 μL Buffer 4

- 100 μL Total

2. Incubate 37C 5hr with occasional mixing

3. Alternative way a. Incubate 37C overnight with occasional mixing

a. Add 10 μL Alu1 b. Incubate 37C 5hr with hourly mixing

This double digestion leads to more complete digestion, but longer digestion might degrade chromatins.

Day3 Ligation and crosslink reversal

1. Add 10μL 10% SDS (stop further digestion) and incubate 65C 20min

2. Prepare ligation ○ 197 μL dH20 ○ 400 μL 2X Quick Ligase buffer ○ 80 μL 10% Triton-X (sequester SDS by forming micells) ○ 8 μL 10mg/mL BSA

a. Mix and incubate room temperature 15min: Sequester SDS with Triton-X ○ 5 μL Quick ligase

○ 800 μL total

Optional: Sequester SDS prior to ligation (preferred) i. Add 610μL dH20 and 80μL 10% Triton-X ii. Centrifuge samples max speed 20min room temp iii. Resuspend pellet in 100μL 10mM Tris-Hcl (pH 7.5) iv. Add 1) 400μL 2X Quick ligase buffer 2) 295μL dH20 3) 5μL Quick ligase

4) 800 μL total

To me these two method does not yield noticeable difference

3. Incubate 2hr room temp

4. Add 10μL 10μg/μL RNAseA 5. Incubate 37C 30min

6. Add 5 μL 20mg/ml Proteinase K 7. Incubate 8hr or overnight 65C

Day4 DNA isolation

1. Pull reactions into 15ml tube

1. Add 1 volume Phenol-Chloroform (optional: add dH20 upto 7ml or 700μL) Final desired volume is 4ml or 400μL for EtOH precipitation 2. Spin down 5min max 3. Collect aqueous layer 4. Repeat 3-5

5. Add 1 volume Chloroform 6. Spin down 5min max 7. Collect aqueous layer

8. Add 0.1 volume 3M sodium acetate pH5.2 and vortex 9. Add 3μL/800μL 20mg/ml glycogen 10. Add 2.5-3 volume ice-cold 100% EtOH 11. Incubate -80C 30min

12. Spin down max speed 20min 4C 13. Resuspend with 1ml cold 70% EtOH and move into 1.5ml eppi tube 14. Spin down max 15min 4C 15. Airdry

16. Resuspend with 500μL TE 17. Add 125μL 4M NH4OAc and 625μL Isopropanol 18. Incubate room temp 30min

21. Spin down max 30min 4C 22. Resuspend with 1ml 70% EtOH 23. Spin down max 20min 4C 24. Airdry

25. Resuspend in TE