Nascent Chromatin Capture. The NCC protocol is designed for SILAC-based massspectrometry

Transcription

1 Extended experimental procedure Nascent Chromatin Capture. The NCC protocol is designed for SILAC-based massspectrometry analysis of nascent versus mature chromatin composition (1). It requires a large amount of cells, which we achieve by using Hela S3 cells in suspension. We used cells labeled by heavy isotopes for purification of nascent chromatin, whereas cells grown in light isotopes were for mature chromatin. For each condition 5 x 10 8 labeled cells were used as starting material (2). I. SILAC labeling and synchronization 1. Label and expand cells in light or heavy SILAC medium in spinner flasks for 9 generations, ending up with 5 x 10 8 cells for each condition. NB: Synchronization of the light culture is started two hours before the heavy culture in order to harvest nascent and mature samples at the same time. 2. Count light cells and adjusts the concentration to 1.1 x 10 6 cells per ml in a total volume of 450 ml light SILAC medium (3). Add 50 ml of freshly made 20 mm thymidine dissolved in light SILAC medium and leave for 17 hours in incubator. 3. Two hours later, count heavy cells and adjusts the concentration to 1.1 x 10 6 cells per ml in 450 ml heavy SILAC medium. Add 50 ml of freshly made 20 mm thymidine dissolved in heavy SILAC medium and leave for 17 hours in incubator. 4. To release cells from the thymidine block, wash thoroughly light and heavy-labeled cells twice with 250 ml of pre-warmed PBS (4) and resuspend cells in 500 ml prewarmed SILAC medium containing 24 M deoxycytidine. Allow cells to progress in S phase for 3 hours in incubator. II. d-utp labeling 1. Count light cells (5,6) and spin 5 x 10 8 cells for 6 min at 300 g at room temperature (RT).

2 2. Remove the supernatant and resuspend carefully in 50 ml of pre-warmed KBH buffer (7). 3. Spin cells for 6 min at 300 g at RT. 4. Remove the supernatant carefully (avoid disturbing the pellet) and resuspend in 10 ml of pre-warmed KBH buffer containing 500 l of biotin-dutp. Incubate 5 min at 37 C in a water bath with continuous and gentle shaking to prevent cells from sedimenting (8). 5. Resuspend cells in 500 ml of light SILAC medium and incubate at 37 C for 20 min (9). 6. Spin cells for 6 min at 300 g at RT. Remove the supernatant, resuspend the pellet in 500 ml of light SILAC medium and transfer to a spinner flask for 2 hours incubation at 37 C (10). 7. Process the heavy cells as described from step II.1 to II.5 using heavy SILAC medium instead of light. NB: Step II.7 should be finished at the exact same time as the incubation time in step II.6 ends. III. Fixation and nuclear extraction NB: From this step, heavy and light labeled cells are processed in parallel. 1. Spin heavy and light cells for 6 min at 300 g at RT. Remove the supernatants and resuspend each cell pellet in 50 ml of cross-linking solution. Incubate immediately on a shaker at 36 rpm for 15 min at RT. 2. To stop cross-linking, add 5 ml of 10% glycine and incubate 5 min at RT on a shaker at 36 rpm. 3. Spin cells for 10 min at 1000 g at 4 C. NB: From this step, work on ice.

3 4. Wash each cell pellet twice in 50 ml of ice-cold PBS-PMSF. 5. Resuspend in 5 ml ice-cold sucrose buffer, and bring the volume to 50 ml. 6. Spin for 10 min at 1000 g at 4 C, resuspend each sample in 75 ml ice-cold sucrose buffer and transfer to a 15 ml dounce homogenizers on ice. 7. Dounce 20 times with tight pestle. Pool the dounced material in a new falcon tube and spin for 10 min at 2500 g at 4 C. 8. Discard the cytoplasmic supernatant (11) and resuspend the nuclear pellet in 50 ml ice-cold glycerol buffer. 9. Spin for 10 min at 2500 g at 4 C and resuspend the nuclear pellet into one volume of ice-cold glycerol buffer. Snap-freeze in liquid nitrogen and store at -80 C until use. This is the nuclear fraction. IV. Preparation of chromatin 1. Thaw the nuclear fraction on ice and resuspend into 10 ml of ice-cold sonication buffer. 2. Spin for 10 min at 2500 g at 4 C. Resuspend the nuclear pellet in sonication buffer, 10 ml for cells. Aliquote into ml tubes and sonicate in a Diagenode Bioruptor at 4 C. Bioruptor setting: High, 28 cycles of 30 sec sonication and 90 sec pause. 3. Spin for 15 min at 5000 g at 4 C and transfer the supernatant (solubilized chromatin) into a new tube. Proceed directly to V or keep at 4 C up to one week (12). V. Affinity purification of biotin-tagged chromatin 1. Dilute solubilized chromatin 1:1 with Dilution buffer (13). 2. Save 100 L of the input. Keep at -20 C. 3. Add 250 L of MyC1 Streptavidin beads and rotate end-over-end over night at 4 C.

4 4. Place tubes on a magnetic stand. Collect the unbound fraction and keep on ice until step V.9. (14) 5. Wash the beads 5 times in 1 ml wash buffer by inverting the tubes 10 times. 6. In the last wash, mix the beads from nascent and mature samples (15) and remove the supernatant carefully. 7. Add 120 l of LSB to the beads and boil for 40 min at 99 C, including a brief vortex and short spin every 10 min to prevent the beads from drying. 8. Place the tube on a magnetic stand and collect the supernatant containing the purified chromatin. Store at -20 C. 9. To improve the yield, process the unbound fraction of step V.4 for a second round of pull-down according to step V.3 to V Add the supernatant from step V.8 and boil the beads as in step V Place the tube on a magnetic stand and collect the supernatant containing the final purified chromatin prep. Store at -20 C until use. VI. Analysis of isolated proteins using mass spectrometry. 1. Run the purified chromatin on SDS-PAGE in a pre-cast Bis-Tris gradient gel 4-12% 1.5 mm (Invitrogen). 2. Stop the electrophoresis, as proteins have entered the gel when the blue color is 1 mm below the well (16). 3. Stain the gel in colloidal blue, cut the band and process for mass spectrometry analysis. VII. Reagents and buffers All reagents should be freshly made. KBH buffer 50 mm KCl 10 mm Hepes (use either Hepes powder or Hepes stock solution with non-adjusted ph)

5 biotin-dutp (IBA tagnology) The DNA labeling is more efficient when the biotin-dutp is fresh since biotin-dutp is unstable in solution. We therefore utilize single-use aliquot of lyophilized biotindutp. 10 min prior to use, briefly spin the lyophilized biotin-dutp and resuspend in 10 mm Tris ph 8 to obtain a 100 nm biotin-dutp solution. Mix well and incubate on ice until use. Cross-linking solution. Just before use, dilute 2 ml of 37% formaldehyde (F8923 Formaldehyde solution methanol stabilizer from Sigma) in 35 ml of PBS. Glycine 10% Prepare 10% glycine (50046 from Sigma, bio-ultra for mol. Biology) solution in autoclaved water at RT. Use at 1% final concentration. PBS-PMSF Just before use, prepare a 1 mm PMSF solution final in ice cold PBS. Keep on ice. Sucrose buffer 0.3 M Sucrose 1% Triton X mm MgOAc Glycerol buffer 25% Glycerol (G5516 from Sigma) 0.1 mm EDTA 0.1 mm EGTA 5 mm MgOAc Sonication buffer 100 mm NaCl 2 mm EDTA ph 8 1 mm EGTA ph 8 0.2% SDS 0.1% Sodium Sarkosyl Prior to use add PMSF to 1 mm final concentration. Dilution buffer 100 mm NaCl 2 mm EDTA ph 8 1 mm EGTA ph 8 Prior to use add PMSF to 1 mm final concentration. Wash buffer

6 100 mm NaCl 2 mm EDTA ph 8 1 mm EGTA ph 8 0.1% SDS Prior to use add PMSF to 1 mm final concentration. VIII. Notes 1. This set-up can be modified to address chromatin maturation at later times or replication stress responses. 2. For western blot analysis, we use 1 x 10 8 cells per sample. 3. We take 0.5 x 10 6 cells from the heavy and light cultures to monitor the asynchronous cell cycle by FACS analyze. 4. We take 0.5 x 10 6 cells from the heavy and light cultures to confirm efficient G1-S arrest by FACS analyze of the cell cycle profile. 5. To start with exactly the same amount of cells, re-count cells after the release from thymidine. 6. We take 0.5 x 10 6 cells from the heavy and light cultures to confirm efficiency of the release from the thymidine arrest by FACS. This is important since impaired thymidine release affect the biotin-dutp labeling 7. It is important to keep the KBH buffer at 37 C, as it increases the biotindutp incorporation efficiency. 8. Incorporation and labeling of biotin-dutp is most efficient in a water bath compared to an incubator. 9. This is the labeling of newly replicated DNA with biotin-dutp. 10. This is the chase to label mature biotin-dutp tagged chromatin. 11. Keep the supernatant if you also want to analyze the cytosolic material. 12. To check the fragment size, take 50 l of soluble chromatin and add 500 l of 10 mm Tris-HCl ph 8. Shake over night at 65 C 750 rpm to de-crosslink chromatin. Then process as for classic phenol/chloroform DNA extraction. 13. This step is important to reduce the SDS concentration, which can affect the binding of biotin to streptavidin. 14. Pool the beads into 4 2ml tubes before washing, by transferring the suspension of beads into each tubes in two rounds. 15. To analyze samples by western blot, keep the two samples separated until the end. 16. For western blot analyze the samples by standard conditions for electrophoresis and proceed with immunoblotting.