Performed using the TruSeq ChIP Sample Prep Kit Cat#IP-202-1012 setA / IP-202-1024 setB
Perform End Repair NOTE: Adjust the concentration of the ChIP enriched DNA ~10ng/50ul in nuclease free water. NOTE: Prepare a fresh stock of 80% ETOH. NOTE: Remove the AMPure XP Beads from 4°C when performing the 30°C incubation and warm to RT° for 30 minutes. NOTE: Remove Resuspension Buffer and End Repair Mix from -20°C and thaw at RT°, place on ice. 1. Prepare the following reaction mix in a 0.2ml PCR plate on ice for a single sample: 50ul ~10ng ChIP Enriched DNA 10ul Resuspension Buffer 40ul End Repair Mix 100ul Total volume 2. Mix well by pipetting 10x and seal plate with a Microseal B plate seal. 3. Incubate at 30°C for 30 minutes in a thermal cycler using the following profile: 30°C
Hold
CHIP30
NOTE: Use heated lid
4. Vortex the AMPure XP Beads until they are fully dispersed. 5. Add 160ul of AMPure XP Beads to the reaction mix. Pipette to mix 10x. Return AMPURE XP Beads to 4°C. 6. Incubate the mixture at RT° for 15 minutes. 7. Transfer reaction plate to the Ambion’s peg style magnetic stand and clear mixture at RT° 15 minutes or until liquid is fully cleared. 8. Remove and discard 127.5 ul of the supernatant from each clarified sample. DO NOT DISTURB BEADS. 9. Repeat step 8 once. 10. Keep the PCR plate on the magnetic stand and add 200ul of freshly prepared 80% ETOH to each well without disturbing beads. 11. Incubate plate at RT° 30 seconds. 12. Remove all of the ETOH from each well without disturbing the beads. Ensure that all ETOH has been removed. 13. Repeat steps 10-12 for a total of two ETOH washes. 14. Leave the plate on the magnetic stand and incubate at RT° for 15 minutes to dry residual ETOH. 15. Remove the plate from the magnetic stand and resuspend the bead pellet in 17.5ul of Resuspension Buffer by pipetting 30x’s. 16 Incubate the plate at RT° for 2 minutes. 17. Place the plate on the magnetic stand at RT° for 5 minutes. 18. Pipetting very slowly transfer 15ul of the cleared supernatant to a new 0.2ml PCR plate.
Safe Stopping Point: Seal plate with adhesive seal and store at -20˚C.
Adenylate 3’ Ends 1. Prepare the following reaction mix in the following order. Gently pipette 10x to mix. 15ul End Repaired DNA 12.5ul A-Tailing mix 2.5ul Resuspension Buffer 30ul Total volume 2. Seal Plate with a Microseal B plate seal.
3. Incubate in a thermal cycler using the following profile:
37°C 70°C 4°C
2
CHIP37
30 min 5 min hold
4. Remove the reaction plate from the thermal cycler when 4°C is reached and place on ice. Proceed to Ligate Adapters.
Ligate Adapters NOTE: Remove appropriate RNA Index Adapters from -20°C and thaw at RT°. Place on ice. NOTE: Remove Stop Ligation Buffer and thaw at RT°. Place on ice. NOTE: Remove AMPure XP Beads from 4°C and warm to RT° for 30 minutes. 1. Briefly centrifuge the thawed RNA Adapter Indexes and Stop Ligation Buffer tubes. 2. Immediately before use, remove the Ligation Mix tube and place on ice. 3. Prepare the ligation reaction on ice in the following order: 30ul 2.5ul 2.5ul 2.5ul 37.5ul
Adenylated DNA Resuspension Buffer Ligation Mix RNA Adapter Index Total volume
NOTE: Remove Ligation Mix from -20°C immediately before use and return to -20°C immediately after use.
4. Gently pipette 10x to mix on ice. Seal plate with Microseal B plate seal. Centrifuge plate 1 min at 280xg. 5. Incubate the PCR plate in a thermal cycler at 30°C for 10 minutes using the following profile: 30°C
Hold
CHIP30
NOTE: Use heated lid
6. Remove plate from cycler and add 5ul of Stop Ligation Buffer to each reaction. Pipette 10x to mix. 7. Vortex the pre-warmed AMPure XP Beads ensuring full dispersal. 8. Add 42.5ul of AMPure XP Beads to each reaction. Pipette 10x to mix thoroughly. 9. Incubate the binding reaction at RT° for 15 minutes. 10. Place the plate on the magnetic stand at RT° for 5 minutes to clarify the samples. 11. Remove and discard 80ul of supernatant from each sample. DO NOT DISTURB BEADS. 12. With the plate remaining on the magnetic stand, add 200ul of freshly prepared 80% ETOH to each sample. 13. Incubate at RT° 30 seconds then remove all of the supernatant without disturbing beads. 14. Repeat steps 12 and 13 for a total of two ETOH washes. 15. Leave the plate on the magnetic stand and incubate at RT° for 15 minutes to dry residual ETOH 16. Remove plate from magnetic stand and resuspend the bead pellet with 52.5ul of Resuspension Buffer. Pipette 30x to mix well. 17. Incubate the plate at RT° for 2 minutes. 18 Place the plate on the magnetic stand at RT° for 5 minutes. 19. Transfer 50ul of cleared supernatant to a new PCR plate. 20. Vortex AMPure XP Beads to mix and add 50ul of beads to each sample for a second clean up. Pipette 10x to mix thoroughly. 21. Incubate the plate at RT° 15 minutes. 22. Place the plate on the magnetic stand at RT° for 5 minutes. 23. Remove and discard 95ul of supernatant from each sample. DO NOT DISTURB BEADS. 24. Keep the PCR plate on the magnetic stand and add 200ul of freshly prepared 80% ETOH to each well without disturbing the pellet. 25. Incubate the plate at RT° for 30 seconds. 26. Remove all of the ETOH from each well without disturbing the beads. Ensure all ETOH is removed. 27. Repeat steps 24 – 26 for a total of two ETOH washes.
3
28. Leave the plate on the magnetic stand and incubate at RT° for 15 minutes to dry residual ETOH. 29. Remove the plate from the magnetic stand and resuspend each bead pellet in 32.5ul Resuspension Buffer. Pipette 30x to thoroughly resuspend beads. 30. Incubate plate at RT° 2 minutes. 31. Transfer plate to magnetic stand and incubate at RT° for 5 minutes. 32. Pipetting very slowly transfer 30ul of clarified supernatant to an appropriately labeled 1.5 ml microcentrifuge tube.
Safe Stopping Point: Store samples at -20˚C.
Purify and Size Select the Ligation Products Perform the size selection using the Sage Pippin Prep NOTE: Bring Pippin Prep loading solution to RT˚. Prepare DNA Samples for loading 1. Initiate the Pippin Prep instrumentation allowing software to launch. 2. Add 10ul of RT˚ loading solution to each sample for a total of 40ul. Mix well and centrifuge briefly.
Program Protocol 1. From main screen select “Protocol” to go to the Protocol Editor Screen. Press “New” to open a new protocol. 2. Select the appropriate cassette type ( 2% Marker B No Overflow Detection). 3. Select a reference lane which will contain the DNA marker. NOTE: Verify lane assignment of software display vs cassette lanes. 4. Enter - bp target size 275bp -bp start size 250bp NOTE: Cut size may need to be adjusted to target optimal size distribution of fragmented sample. -bp end size 300bp 5. Enter sample name into the “Sample ID” field. 6. Select “Save as” and name protocol to retain for future applications (TruSeq ChIP 250-300bp). 7. Return to Main Screen.
Prepare Cassette 1. Remove cassette from packaging and wipe off excess moisture. Verify correct cassette type is used. 2. Visually inspect buffer chamber and gel columns. From side view ensure buffer chamber volume is more than ½ full. Inspect gel column for breakage, bubbles or gaps. Any lanes with these defects must be abandoned. Remaining lanes are still functional. 3. Inspect for air gaps in and behind elution wells by tilting cassette to the left (loading well end). Tap on bench lightly to dislodge. 4. Install cassette into tray of Pippin Prep with loading wells to the left. 5. Remove sealing tape without tipping the cassette. 6. Remove all buffer from elution well and replace with 40ul of electrophoresis buffer. 7. Test electrophoretic current continuity: a. Close sliding lid on instrument. b. In the run screen select “Manual Mode” from the “Protocol Name” drop down menu. c. Press “Test” in controller on the Main Screen. Test takes 30 sec. finishing with “Pass” of “Fail” message. d. If test fails – in the separation lane: lane unusable – no recovery - in the elution lane: Verify 40ul of running buffer is present in the elution well and re-test. If it fails again lane can be used for reference sample. 7. Cover all collection wells with tape to minimize recover volume to ~50ul. Using razor blade cut tape between elution wells so that individual tape segments can be remove to minimize potential cross contamination.
4 Sample Loading 1. Verify that sample wells are completely full. Top off wells with electrophoresis buffer if necessary. Total well volume is 70ul. 2. Remove 40ul of running buffer from the loading well leaving ~30ul in well. 3. Load 40ul of pre-mixed Pippin Prep DNA Marker into the sample lane that has been assigned for the reference. (Reference lane can be changed as needed). 4. Load 40ul of the supplemented sample into the appropriate lane loading well. 5. Close lid 6. Select desired protocol from the “Protocol Name” drop down menu. (ie: TruSeq ChIP 250-300bp) 7. Press “Start”.
Sample Collection 1. When complete, remove elution well tape and transfer all sample from sample elution well to a labeled microcentrifuge tube. 2. Determine volume of collected sample. Volume can be from 40-70ul. 3. Clean electrodes of Pippin Prep by installing cleaning cassette filled with H2O and closing lid for 30 seconds. Remove Cassette.
Sample Clean-Up Use the Qiagen MinElute PCR Purification Kit (cat# 28004) for clean-up procedure. Perform the PCR Purification Protocol. 1. Add 5 volumes of PB buffer (with Indicator) to 1 volume of the Pippin Prep elute (250ul PB+I buffer + 50ul Pippin Prep Elute). 2. Check the color of the mixture and ensure that it is yellow (similar to the original color of buffer ERC). a. If color is orange or violet add 10ul of 3M sodium acetate pH 5.0 and mix. The color will return to yellow. 3. Place a MinElute column in a 2ml collection tube. 4. Bind DNA by adding the sample to the MinElute column and centrifuging 1 min at 13,000 RPM at RT˚ 5. Discard the flow through and place the MinElute column back in the same collection tube. 6. Wash column by adding 750ul of Buffer PE to the MinElute column and centrifuge 1 min at 13,000 RPM at RT˚ 7. Discard flow-through and place MinElute column back in the same collection tube. 8. Centrifuge MinElute column for an additional 1 minute to remove any residual wash buffer PE. 9. Place the MinElute Column in a new labeled 1.5ml microcentrifuge tube. 10. Elute DNA by adding 23ul of EB buffer (10mM Tris-HCl pH 8.5) to the center of the membrane. Let column stand at RT˚ 1 minute. 11. Centrifuge MinElute column for 1 minute at 13,000 RPM. 12. Check volume of eluted samples and verify that 20ul+ are obtained. Add resuspension buffer, if needed, to obtain 20ul.
Safe Stopping Point: Store labeled tubes at -20˚C.
Enrich DNA Fragments NOTE: Remove the PCR Master Mix and PCR Primer Cocktail and thaw at RT° then place on ice. NOTE: Remove AMPure XP Beads from 4°C and warm to RT° for 30 minutes. NOTE: Remove Resuspension Buffer from -20°C and thaw at RT°, place on ice. 1. Prepare the following reaction on ice for each sample using a PCR plate. 20ul 5ul 25ul 50ul
Sized DNA PCR Primer Cocktail PCR Primer Master Mix Total volume
5 2. Mix by pipetting 10x. Seal Plate with MICROSEAL “A” (BioRad). 3. Transfer the PCR plate to a thermal cycler and amplify the samples using the following profile:
4. Vortex the AMPure XP Beads until fully dispersed. Return beads to 4°C. 5. Add 50ul beads to each sample and pipette 10x to mix thoroughly. 6. Incubate the plate at RT° for 15 minutes. 7. Transfer plate to magnetic stand for 5 minutes at RT° ensuring that beads have fully cleared. 8. Remove and discard 95ul of supernatant from each sample. DO NOT DISTURB BEADS. 9. Leave plate on magnetic stand and add 200ul of freshly prepared 80% ETOH without disturbing beads. 10. Incubate plate at RT° for 30 seconds then remove and discard all of the supernatant while avoiding beads. 11. Repeat steps 9 and 10 for a total of two ETOH washes. 12. Leave plate on the magnetic stand and incubate at RT° 15 minutes to dry. When complete remove plate from magnetic stand. 13. Resuspend the dry bead pellet with 17.5ul of Resuspension Buffer. Gently pipette 30x to fully resuspend beads. 14. Incubate plate at RT° for 2 minutes. 15. Transfer plate to magnetic stand at RT° for 5 minutes ensuring that sample clarifies. 16. Pipetting very slowly transfer 15ul of each cleared supernatant to an appropriately labeled 1.5 ml microcentrifuge tube.
Safe Stopping Point: Store libraries at -20˚C.
Validate Library 1. Run 1ul of each sample on the Agilent Bioanalyzer using the DNA1000 kit. Based on yield of libraries a DNA High Sensitivity Agilent run may be required. 2. Check size, purity and concentration of the sample. NOTE: Library size should be ~ 275bp. 3. Using Agilent values, calculate the nM concentration of each sample using the concentration conversion. 4. Prepare a 2nM stock of each sample using Qiagen’s EB buffer supplemented with 0.1% Tween20.
Pool Libraries (If required) 1. Pool equal volumes of the appropriate 2nM aliquots into properly labeled 1.5ml microcentrifuge tubes to obtain multiplexed samples. 2. Proceed to cluster generation and sequencing.
