HISTONE CHIP PROTOCOL

Elution, Decross-Linking And DNA Purification

• After removing the last wash buffer, add 100 μL of elution buffer A to the beads and incubate for 30 min under constant rotation at room temperature.
• Resuspend the beads pellet and transfer it into a new 200 μL tube.
• Briefly spin the tubes and place them into a magnetic rack.
• Transfer the supernatant to a new tube and add 4 μL of elution buffer B. Also add 97.5 μL buffer A and 4 μL of buffer B to the 2.5 μL input sample. Incubate for 4 hours or overnight at 65°C.
• Pool samples if necessary.
  Note: Up to two samples can be easily pooled. If more than two samples need to be pooled, process each sample purification individually, pool final eluates at the end of the magnetic bead purification and concentrate.
• Add 2 μL of carrier to each IP and input sample.
• Add 100 μL of 100% isopropanol to each IP and input sample.
  Note: Following the addition of isopropanol the solution may become cloudy. This is not detrimental to your experiment and will not influence the quality or quantity of your purified DNA.
• Resuspend the high-DNA recovery magnetic beads and transfer 10 μL to each IP and input sample (Final volume should be 116 μL per reaction).
• Note: Keep the beads in liquid suspension during storage at 4°C and at all handling steps, as drying will result in reduced performance.
• Incubate IP and input samples for 10 min at room temperature on a rotating wheel (40 rpm).
• Prepare the wash buffer 1 containing 50% isopropanol (for 24 reactions) by mixing 2 mL wash buffer 1 w/o isopropanol and 2 mL isopropanol (100%). Never leave the bottle open to avoid evaporation.
• Briefly spin the tubes, place them in a suitable magnetic rack for 0.2-mL tubes, wait 1 min and discard the buffer.
• Add 100 μL wash buffer 1 per tube. Close the tubes and vortex well until the beads pellet is completely broken.
  Note: In order to avoid the beads pellet to be too difficult to break down, do not let the beads for too long on the magnet, incubate for 30 sec at room temperature on a rotating wheel (40 rpm). Always briefly spin the tubes to bring down liquid caught in the lid prior to positioning into the magnetic rack.
• Prepare the wash buffer 2 containing 50% isopropanol as follows (for 24 reactions) by mixing 2 mL wash buffer 2 w/o isopropanol and 2 mL isopropanol (100%). Never leave the bottle open to avoid evaporation.
• Briefly spin the tubes, place in a suitable magnetic rack for 1.5-mL tubes, wait 1 min and discard the buffer.
• Add 100 μL wash buffer 2 per tube. Close the tubes and vortex well until the beads pellet is completely broken.
  Note: In order to avoid the beads pellet to be too difficult to break down, do not let the beads for too long on the magnet, incubate for 30 sec at room temperature on a rotating wheel (40 rpm). Always briefly spin the tubes to bring down liquid caught in the lid prior to positioning into the magnetic rack.
• Briefly spin the tubes and place them a suitable magnetic rack for 0.2-mL tubes, wait 1 min and discard the buffer.
• Spin the tubes again and place them on the magnetic rack. Discard the remaining wash buffer 2 if necessary.
• Resuspend the beads pellet with 25 μL of elution buffer C. Incubate at room temperature for 15 min on a rotating wheel (40 rpm).
• Spin the tubes and place them into a suitable magnetic rack for 1.5-mL tubes, wait 1 min and transfer the supernatants into a new labelled 1.5 mL tube and discard the beads.
• Place the DNA on ice and proceed to any desired downstream applications or store it at -20°C or -80°C until further use.
• Take 5 μL (10%) of immunoprecipitated DNA and determine the concentration with, for example, the Quant-iT High Sensitivity dsDNA Assay Kit and Qubit fluorometer.
• Store the DNA at -20°C until you are ready to analyze it with qPCR or by high throughput sequencing.
• Note: The elution buffer C is suitable for direct qPCR analysis, whole genome amplification, chip hybridization and Next-Generation sequencing.

ChIP-Seq Protocol

This protocol has been optimized forchip-seq histone modificationl on an Illumina HiSeq sequencer. However, other sequencing systems such as the Illumina MiSeq or the Thermo Fisher SOLiD systems can also be used. Refer to their sequencing protocols for the generation of sequencing data.