Generation af indfødte kromatin Immunoprecipitation sekventering biblioteker for Nucleosome tæthed analyse

The overall goal of this procedure is to generate native chromatin immunoprecipitation sequencing libraries for nucleosome density analysis. This method can answer questions in epigenetics such as revealing epigenomic features in a cell population at a single nucleosome level and resolving heterogeneous chromatin signatures into their continuous elements. The main advantage of this technique is to utilize MNase property of preferential access to open chromatin region to generate a measurement that combines MNase accessibility with histone modification.

Generally, individuals new to this method will struggle because they are not able to obtain optimal MNase digestion. Begin this protocol with cell preparation as described in the text protocol. On day one, thaw each cell pellet in a 37-degrees-Celsius water bath for 10 seconds.

Then, transfer the cells to ice. To each cell pellet, immediately add an ice-cold one-X lysis buffer plus one-X PIC to a final concentration of 10, 000 cells per 20 microliters. Mix 10 times by pipetting up and down without creating bubbles.

Working on ice, aliquot 20 microliters per well of the resulting lysates into a 96-well plate. Cover the plate with the plastic seal before incubating on ice for 20 minutes. Label the plate as MNase and record the wells to a template key.

Just before 20-minute lysis is complete, dilute the MNase one enzyme with MNase one dilution buffer to a final concentration of 20 units per microliter and keep it on ice. Working on ice, prepare the MNase one digestion master mix as described in the text protocol. Then, aliquot 20 microliters of the master mix per each row of samples, plus five microliters of extra volume into a 96-well reservoir plate.

After the lysates finish incubating, remove the MNase digestion plate from the ice. Using a multi-channel pipette, add 20 microliters of MNnase one digestion master mix into each row of samples and mix by pipetting up and down 10 times. Change the tips between rows.

Then, incubate the plate at room temperature for exactly five minutes. To stop the reaction after five minutes, use a multi-channel pipette to add six microliters of 250-micromolar EDTA into each row of samples and mix up and down a few times. Make sure to change tips between rows.

After EDTA addition, switch the setting of the pipette to 20 microliters, and mix 10 times as before to assure complete stop of the digestion reaction. Finally, add six microliters of 10X lysis buffer to each row of the MNase digested samples and mix well by pipetting up and down 10 times. Cover the plate with a plastic seal and incubate on ice for 15 minutes.

Prepare the antibody bead complex plate and the pre-clearing plate as described in the text protocol. Quick-spin both plates by centrifuging for 10 seconds at 200 times G.Then, place the antibody bead complex plate on a plate magnet and wait 15 seconds for the solution to become clear. Carefully remove and discard the supernatant using a pipette without disturbing the beads.

Then, remove the plate from the plate magnet and keep it on ice. Place the pre-clearing reaction plate on a plate magnet and wait 15 seconds for the beads to separate and for the solution to become clear. Without disturbing the beads, use a pipette to carefully transfer the supernatant to the corresponding wells of the antibody bead complex plate kept on ice.

Mix each well gently by pipetting up and down 15 times. Seal the plate well with an aluminum plate cover and incubate overnight at four degrees Celsius on a rotating platform. Following incubation, relabel the plate IP Reaction.

On day two, set the heating mixer to 65 degrees Celsius, then prepare a low-salt wash buffer and high-salt wash buffer on ice. Quick-spin the IP reaction plate for 10 seconds at 200 times G.Place the IP reaction plate on a plate magnet and wait 15 seconds for the solution to become clear. Using a multi-channel pipette, without disturbing the beads, carefully remove and discard the supernatant.

Take the plate off the plate magnet and place it on ice. To each row of samples in the IP reaction plate, add 150 microliters of ice-cold low-salt wash buffer and mix slowly up and down for 10 times to fully resuspend the beads. Place the IP reaction plate back onto the plate magnet and remove the supernatant as before.

Then, place the plate back on ice and repeat the wash steps for a total of two washes. Working on ice, add 150 microliters of the high-salt wash buffer to each row of samples in the IP reaction plate. Mix the samples slowly by pipetting up and down 10 times to fully resuspend the beads.

After removing the supernatant as before, place the IP reaction plate on ice and prechill a new 96-well plate beside it. To each row of samples in the IP reaction plate, add 150 microliters of the high-salt wash buffer and mix slowly 10 times by pipetting up and down to fully resuspend the beads. After resuspension, transfer each row of samples to the corresponding row of the new, pre-chilled 96-well plate.

Discard the old plate. Place the new IP reaction plate on the plate magnet and discard the supernatant as before. Keep the plate at room temperature.

To each row of samples in the IP reaction plate, add 30 microliters of the ChIP elution buffer and mix slowly by pipetting up and down 10 times. Take care to prevent bubble formation. Seal the plate with a PCR cover and incubate in a heating mixer at 65 degrees Celsius for 1 1/2 hours with a mixing speed of 1, 350 RPM.

After a 1 1/2-hour incubation, spin down the IP reaction plate at 200 times G for one minute at four degrees Celsius. Then, place the IP reaction plate on a plate magnet and wait for the solution to clear. Using a multi-channel pipette, without disturbing the beads, carefully transfer 20 microliters of the supernatant into a new 96-well plate using fresh tips for each row.

Label the plate IP Reaction and keep it at room temperature. Proceed to protein digestion followed by library construction as described in the text protocol. Shown here are representative results of optimal, under-digested and over-digested MNase digested chromatin before library generation.

The optimally-digested profile is dominated by single nucleosome fragment sizes while not over-digested to allow for recovery of higher order nucleosome fragments. Suboptimal digestion of chromatin will also be apparent in the profile of the sequencing library generated from the IP material. To validate the libraries by qPCR, fold enrichment of IP libraries with respect to input library was assessed for positive and negative targets.

In addition, inspection of the aligned reads of libraries with high qPCR assessed fold enrichment on a genome browser should reveal visually detectable enrichments as compared to the input library. Successful nucleosome density ChIP-sequencing libraries will contain highly-correlated replicates with a significant portion of aligned reads within MACS2-identified enriched peaks. Once mastered, this technique can be done in two days if it is performed properly.

After watching this video, you should have a good understanding of how to generate native chromatin immunoprecipitation sequencing libraries for nucleosome density analysis. While attempting this procedure, it is important to remember to make sure that the chromatin is optimally digested and only use antibody that show a high affinity with the epitope of interest and show little or no cross-reactivity with other epitopes. Following this procedure, computational modeling of MNase accessibility can be performed to answer questions such as epigenetic signatures due to heterogeneity within a cell population.

This method provides insight into combinatorial effect of nucleosome position and local density as well as post-translational modification of their histone subunits and it can be applied to any systems such as human or mouse, primary cells and frozen tissues.