Luban Lab ChIP-seq protocol

Transcription

1 Luban Lab ChIP-seq protocol (updated by Anetta Nowosielska, December 2016) University of Massachusetts Medical School Program in Molecular Medicine 373 Plantation Street, Biotech 2 suite 319 Worcester, MA Lab members: Ann Dauphin, William Diehl, Smita Jaiswal, Kyusik Kim, Sean Matthew McCauley, Patrick McDonel, Anetta Nowosielska, Leonid Yurkovetskiy, Yetao Wang PI: Jeremy Luban 1. Cell Preparation, Crosslinking And Nuclei Isolation (truchip chromatin shearing reagent kit, Covaris Cat. No ) KIT CONTENTS Buffer A 10X Fixing Buffer Buffer B 5X Lysis Buffer Buffer C 10X Wash Buffer Buffer D3 10x SDS Shearing Buffer (1X Buffer D3 composition: 0.1% SDS, 15 mm Tris ph7.6, 1mMEDTA ) Buffer E 1X Quenching Buffer Buffer F 100X Protease Inhibitor cocktail 1. Prepare solutions for the appropriate number of samples being processed fresh before starting. 1X cold PBS 1X Fixing Buffer A Final Volume: 2.0 ml per sample -Store on ice Final Volume: 0.5 ml per sample -Mix 50 of Fixing Buffer A with ml Water

2 Fresh 11.1% Formaldehyde Final Volume: 1 ml per 1 to 20 samples -Mix 690 of 16% Fresh Formaldehyde with 310 Water 9. Collect cells by centrifugation at 200 x g for 5 minutes, 4 C. 10. Proceed to nuclei preparation and chromatin shearing steps. NOTE: It may be possible Quenching Buffer E Place in a 55 C water bath to dissolve crystals, then place at ambient NOTE: The use of fresh methanol-free formaldehyde is essential to achieve reproducible results. NOTE: Use Molecular Biology Grade Water for the preparation of all solutions. 2. Collect cells by centrifugation at 200 x g for 5 minutes, room temperature. Remove media and wash cells once with PBS and collect cells again by centrifugation. PBS 400 Input cell number 1 3 x 106 Cells Centrifuge Tube 2.0 ml 3. Re-suspend cells in room temperature Fixing Buffer A. Fixing Buffer A Crosslink cells by adding freshly prepared 11.1% formaldehyde solution to a final concentration of 1% and start timing the crosslinking reaction. Fresh 11.1% Formaldehyde Place cells on a rocker at room temperature for 5 minutes to allow for efficient crosslinking. 6. Quench the crosslinking reaction by adding the appropriate volume of Buffer E to the fixed cells. Keep cells on a rocker at room temperature for an additional 5 minutes. Quenching Buffer E Collect cells by centrifuging at 500 x g for 5 minutes at room temperature. 8. Aspirate the supernatant and wash twice with cold PBS. Cold PBS 300

3 10. Proceed to nuclei preparation and chromatin shearing steps. NOTE: It may be possible to flash-freeze the fixed cells in liquid nitrogen at this time and store at -80 C. 11. Prepare the proper number of suspension or adherent cells according to the above protocol. Place the required number of Bioruptor pico microtubes on ice to pre-chill while preparing samples to shear. Number of fixed cells Lo w Cell 1-3 x Prepare the correct volume of fresh solutions for the nuclei preparation and chromatin shearing before use. 1X Lysis Buffer B 1X Wash Buffer 1X Shearing Buffer D3 Final Volume: 0.5mL per sample -Mix 100 5X Lysis Buffer B with 400 water. -Add 5 of 100X Buffer F. -Store on ice. Final Volume: 0.5 ml per sample -Mix 50 10X Wash Buffer B with 450 water. -Add 5 of 100X Buffer F. -Store on ice. Final Volume: 1 ml per sample -Mix X Shearing Buffer D3 with 0.9 ml water. -Add 10 of 100 X Buffer F. -Store on ice. NOTE: Use Molecular Biology Grade Water for the preparation of all solutions. 13. Add Lysis Buffer B containing 1X protease inhibitors to cross-linked cells to lyse plasma membrane, gently resuspend by aspirating/dispensing 4 times. If cells were frozen after formaldehyde fixation, thaw cells on ice first. Lysis Buffer B Transfer to 1.5 ml microcentrifuge tube. 15. Incubate for 10 minutes on a rocker at 4 C 16. Collect intact nuclei by centrifugation at 1,700 x g for 5 minutes, 4 C. Decant the supernatant without disturbing the nuclei pellet. 17. Gently resuspend pellet in Wash Buffer C containing protease inhibitor and incubate on a rocker for 10 minutes at 4 C.

4 Wash Buffer C Collect nuclei by centrifugation at 1,700 x g for 5 minutes, 4 C. Carefully remove and discard the wash solution, taking care not to disturb the nuclei pellet. 19. Gently rinse the sides of the tube with 1X Shearing Buffer D3 containing Protease inhibitor. Slowly dispense the buffer down the entire circumference of the upper-inside of the tube, taking care not to disturb the nuclei pellet. Shearing Buffer D Collect nuclei by centrifugation at 1,700 x g for 5 minutes, 4 C. Decant the supernatant without disturbing the nuclei pellet. 21. Repeat steps 19 and 20 an additional time. Carefully remove and discard the supernatant, taking care not to disturb the nuclei pellet. Resuspend nuclei pellet in the Shearing Buffer D3. Shearing Buffer D Chromatin Shearing 1. Sonicate using Bioruptor pico (Diagenode cat# B ) with 6 cycles of 30 sec ON and 30 sec OFF at 4 C for 1-2 mln cells/sample (for more cells increase cycle number) 2. After shearing centrifuge samples >10,000 x g, 4 C for 5 minutes to pellet insoluble material. 3. Transfer the supernatant to a new pre-chilled microcentrifuge tube; proceed with the immunoprecipitation step. Sheared chromatin can be stored at 4 C for up to 2 days. 4. Chromatin pre-clearing For subsequent immunoprecipitation, sheared chromatin is diluted ChRIPA buffer. ChRIPA Buffer: 1 mm EDTA 0.5 mm EGTA 0.5% sodium deoxycholate (Critical: <4 weeks old) 1% NP40 0.1% SDS Made up in 1X PBS (from 10X stock) 1X proteinase inhibitor cocktail (PIC) (add fresh) 1. Prepare Protein A beads for pre-clearing. Completely resuspend Protein A Dynabead slurry (ThermoFisher Scientific Cat. No 10008D) and transfer 60 ul bead suspension per 1mln cells starting material to a fresh tube(s). Capture on magnet and remove supernatant.

5 2. Wash beads twice with 1 ml ChRIPA. Resuspend and pool all beads into 100 ul aliquots per chromatin sample. 3. Transfer the cleared supernatant(s) from step 8 to each of the tubes with Protein A beads. Rotate (preferably end-over-end) at 4 o C for at least 2h. 4. Capture beads on magnet and transfer each pre-cleared lysate to a fresh LO-Bind tube, measuring the volume of each sample for the yield estimate. Store at 4 C during QC. 5. Testing efficiency of chromatin shearing: 1. Take a 10 aliquot of the sheared sample and transfer to PCR tube. 2. Add 1 of RNase A (10 mg/ml) and incubate at 37 C for 30 min. 3. Add 5 of Proteinase K (NEB) and reverse crosslink by heating at 65 C overnight. 4. Purify DNA using either a commercial column based kit (Zymo Research columns) 5. Elute from column with 15 of elution buffer (10 mm Tris-HCl, ph 8.5). 6. Add 1 volume of loading dye to 5 volumes of purified DNA. 7. Load ng of purified DNA per lane. 8 1 of purified DNA can be analyzed on an Agilent 2100 BioAnalyzer or TapeStation. 6. Immunoprecipitation 1. Save ng prepared chromatin as INPUT sample, store at 4 o C until Elution steps 2. Dilute remaining prepared chromatin in ChRIPA with fresh (1X PIC) such that each IP is >500ul 3. Set up IP reactions in separate 1.5 ml tubes using the following quantities. Amount of Protein A beads is equivalent to 25 ul stock slurry per ug antibody used. Chromatin Antibody source Mark H3K4me1 Diagenode cat#c Lot# A1862D H3K4me3 Millipore (Upstate) cat#05-745r Lot# H3K27ac Diagenode cat#c Lot#A D Chromatin (ug) Antibody (ug) Prot A beads (ul) Incubate on rotator 4h-overnight at 4 o C 5. Meanwhile, block Protein A beads: Transfer 25 ul Protein A Dynabead suspension per ug antibody used to an Eppendorf tube. Capture on magnet and remove sup. Wash beads 2 times with 1 ml blocking buffer. Blocking buffer: 1X PBS 0.5% BSA 0.5% Tween-20 Block beads in at least 1 ml blocking buffer for at least 1h at room temp, or overnight at 4 o C Capture on magnet, remove sup, and resuspend in a convenient volume of ChRIPA buffer for the next step 6. Capture Ab-bound chromatin complexes by adding the appropriate volume of blocked Protein A Dynabeads (equiv to 25 ul original suspension per ug of IgG) directly to the IP reactions. Mix

6 by inversion and rotate for >1h at room temperature. 7. Spin tubes briefly and capture beads on magnet stand(s) for at least 2 min. Open caps and discard supernatant by inverting entire stand over sink. 8. Resuspend beads completely in 180 ul ice-cold ChRIPA buffer (with fresh PIC) and transfer suspensions to strip tubes or plate. Go back and rinse any remaining beads from each tube using 40 ul ChRIPA buffer and collect in the same strip tubes. Collect beads on 96 well magnet. (From this point forward, all washes are carried out on a 96w magnet by adding ul wash buffer with a multichannel pipettor, moving the strips/plate back and forth on the magnet to shift the beads side-to-side in each tube, capturing for 1 min., then removing supernatant with a multichannel) 2. For each sample, prepare a master mix of 3 ul Direct Elution Buffer and 2 ul RNase A (0.5 o 7. Washes 1. Wash beads 5X with ice-cold ChRIPA buffer 2. Wash beads 2X at room temp with RIPA-500 buffer. RIPA-500 buffer: 10 mm Tris-HCl ph8, 1 mm EDTA, 500 mm NaCl, 1% Triton X-100, 0.1% Na-DOC, 0.1% SDS (add last and mix quickly to prevent precipitation do not refrigerate!) 3. Wash beads 2X with ice-cold LiCl wash buffer. LiCl wash buffer: 10 mm Tris-HCl ph8, 1mM EDTA, 250 mm LiCl, 0.5% NP40, 0.5% Na-DOC 3. Wash beads 2X with ice-cold TE buffer. TE buffer: 10mM TrisCl ph 8 1 mm EDTA ph 8 5. After final wash, remove as much supernatant as possible. 8. DNA Elution (include INPUT sample collected at IP Step 1) 1. Remove tubes/plate from the magnet and add 50 ul Direct Elution Buffer (room temp), and mix by pipetting up and down once. Direct Elution Buffer (DEB): 10 mm Tris-HCl ph8 5 mm EDTA 300 mm NaCl 0.5% SDS (add last to avoid precipitation) (Prepare and store at room temp and filter before use)

7 mg/ml). Add 5 ul mix to each reaction, seal tubes/plate and incubate at 37 C for 30 minutes. 3. For each sample, prepare a master mix of 1.5 ul DEB, 2.5 ul Proteinase K (20 mg/ml), and 1 ul glycogen (20 mg/ml). Add 5 ul mix to each reaction, mix by pipetting, seal and place in a PCR machine to incubate 2h at 37 o C, then 4h-overnight at 65 o C. 4. Capture beads on a 96w magnet and transfer supernatant (eluate) to fresh strip tubes/plate. 5. Add 140 ul SPRI beads to 60 ul eluate (2.3X ratio), mix by pipetting 25 times and incubate at room temp for 2 minutes, then capture on magnet for 4 minutes. Carefully remove and discard supernatant do not aspirate off any beads. 6. Leave on magnet and wash beads twice with ul 70% ethanol, without disturbing beads. Be very careful not to remove any beads during washes. After final wash, remove as much ethanol as possible without losing beads. 7. Air dry beads until ethanol is completely evaporated (~4 min). Any residual ethanol can severely compromise yield. 8. Elute samples by adding 10 ul of 10 mm Tris ph8 and resuspending beads by pipetting no less than 20 times. Incubate for 2 minutes, and then place back on magnet for 4 minutes. Transfer supernatant to fresh tubes. 9. For library preparation use Ovation Ultralow System V kit from NuGEN (Cat. No ). CONSUMABLES AND INSTRUMENTS: Molecular Biology Grade Water Thermo Scientific (Cat. No. SH ), Mo Bio (Cat. No ), or equivalent Fresh methanol-free 16% Formaldehyde Thermo Scientific (Pierce) (Cat. No , 10 ml or 28906, 1mL ampules) Phosphate Buffered Salt Solution (PBS) Thermo Scientific (Cat. No. SH30256.FS) RNase A (DNase free) Thermo Scientific (Cat. No. EN0531) Proteinase K (RNase and DNase free) NEB (Cat. No. P8102S) Refrigerated centrifuge having 15,000 x g capability Rocker Nutator or equivalent Bioruptor pico (Diagenode cat# B ) 1.5 ml Bioruptor microtubes with caps (Diagenode Cat. No. C ) 1.5 ml tube holder for Bioruptor pico (Diagenode Cat. No. B ) Water cooler (Diagenode Cat. No. B ) DNA Clean & Concnetrator-5 Capped Columns (ZymoResearch Cat. No D4014)