The overall goal of this procedure is to investigate the binding profiles of different histone modifications, using low-abundance embryonic samples in order to improve the functional annotation of vertebrate genomes. This method can help answer key questions in the developmental biology field such as, which are the key enhancers and genes defining the identity of a particular embryonic tissue. The main advantages of this techniques are that it requires a limited amount of particular material and it can also be used for both local specific and genome wide analysis.
Though this method can provide insights into transcription regulation or cellular heterogeneity with the embryonic tissues, it can also be applied to other systems such as, patent samples. To obtain stage HH19, chicken embryos for this experiment, incubate fertile white Leghorn hen eggs in horizontal orientation at 37 degrees celsius and 80%humidity for three days. To start isolating the HH19 embryos, make them accessible by extracting three milliliters of albumin with a 10 millimeter syringe to lower the blastoderm.
In order to remove extra embryonic membranes, use fine scissors to make a small opening in the eye. Add one milliliter of lock solution. With the help of a bended needle, add 0.5 milliliters of Indian black ink.
Use fine surgical scissors and forceps to cut off the extra embryonic membrane that surrounds the embryo. Then, transfer the embryo with a perforated spoon to a 4.5 centimeter petri dish containing 20 milliliters of PBS solution. Working on ice under a stereo microscope, use fine scissors and forceps to dissect and discard the amnion and the remaining parts of extra embryonic membrane.
Then, cut the transverse spinal neuro tube or SNT segment at the brachial level of the embryo. Extend the dissection caudally into the thoracic region to isolate the SNT. To finish, use forceps to remove the tissues that laterally and ventrally surround the SNT.
After isolating SNTs, immerse each segment in a 3.5 centimeter petri dish containing 37 degree celsius warm trypsin. When loosening of the non-neuro tissues around the SNT is visible under a stereo microscope, transfer the samples into cold 1x PBS. To stop the trypsinization, transfer the samples into a new dish with cold DMEM containing 10%FBS.
Use forceps to manually remove the remaining mesenchymal and ectodermal tissues from the SNT. Transfer this clean SNT section to a 1.5 milliliter tube and flash freeze it in liquid nitrogen. Next, homogenize the tissue according to the text protocol.
Add 13.5 microliters of 37%formaldehyde and incubate at room temperature on a rotator for 15 minutes. To quench the formaldehyde, add 25 microliters of 2.5 molar glycine to the tube and incubate at room temperature on a rotator for 10 minutes. Centrifuge the tube and then, wash the pellet according to the text protocol.
After crosslinking, re-suspend the pellet in 300 microliters of ice cold, complete lysis buffer by pipetting. Incubate the samples at four degrees celsius on a rocking platform for 10 minutes. Next, sonicate the samples at four degrees celsius.
After sonication, centrifuge the samples at 16, 000 x G at four degrees celsius for 10 minutes. Transfer the supernatant or lysate to a fresh 1.5 milliliter tube and discard the cellular debris pellet. Dilute the lysate according to the text protocol.
Then, keep a small aliquot of each sample as input control and freeze at 20 degrees celsius. Incubate the remaining samples that will be subjected to chromatin precipitation or chip with appropriate antibodies according to the text protocol. Then, add 300 microliters of antibody bound chromatin to previously washed magnetic beads.
Invert the tubes to mix the samples. To bind the antibodies to the beads, place the tubes on a tube rotator and rotate vertically at four degree celsius for a minimum of four hours. To wash the chromatin bound beads, add one milliliter of ice cold RIPA wash buffer to the tube kept on ice and mix by inverting or flicking the tube.
After placing the tube into the magnetic holder at room temperature, wait for the beads to settle on the side. Pour off the clear liquid and repeat this wash three more times. After adding one milliliter of TE 50 millimolar sodium chloride to the tube, transfer the chromatin bound beads in this solution to a fresh 1.5 millimeter microcentrifuge tube.
Place the tube into the magnetic holder and when the beads have settled, pour off the liquid. Next, centrifuge the beads at 900 x G at four degrees celsius for three minutes in a pre-cooled centrifuge. After the beads have been settled in the magnetic holder, remove all remaining TE using a gel loading tip.
Working at room temperature, add 210 microliters of elution buffer to the beads and re-suspend by flicking. Incubate the tube in a pre-warmed heat block at 65 degree celsius for 15 minutes, while shaking. After incubation, centrifuge the tubes with the beads at 16, 000 x G at room temperature, for one minute.
Once the beads have settled in the magnetic holder, transfer the supernatant to a fresh microcentrifuge tube. Thaw the previously frozen input control sample from 20 degrees celsius to room temperature. Add 90 microliters of elution buffer to the sample and mix by briefly vortexing.
To reverse the cross-links, incubate the chip and control samples at 65 degrees celsius overnight. Working at room temperature, add one volume of TE buffer per tube and invert to mix. To digest the RNA, add RNAse A to the control and chip samples to a final concentration of 0.2 milligrams per milliliter and invert the tubes to mix.
Incubate the tubes in a pre-warmed heat block at 37 degrees celsius for two hours. To digest the protein for the subsequent DNA purification, add protease K to a final concentration of 0.2 milligrams per milliliters to the tubes and invert the tubes to mix. Incubate the tubes in a pre-warmed heat block at 55 degrees celsius for two hours.
Extract and purify the DNA according to the supplementary materials. This DNA can now be used for quantitative PCR and chip seek library preparation. Sonication efficiency essential for a successful chip is confirmed by agarose gel electrophoresis of the input control samples.
In this example, using chicken spinal neural tube or SNT sections, 11 sonication cycles were used to obtain an optimal fragment size ranging from 200 to 500 base pairs. Chipped DNA was analyzed by quantitative PCR to measure the enrichment levels of activating and repressive histone marks around the promoter regions of genes that are active and inactive in the chicken SNT. This shows that it is possible to evaluate the chipped quality by quantifying only a few low side without losing much DNA for further analysis.
After chip seek libraries were prepared in sequence, a representative locust is shown for H3K27 tri methylation and H3K4 tri methylation chip seek data generated in chicken SNT. Similar chip seek profiles were also successfully generated in different chicken embryonic tissues, such as maxillary prominences and stage HH3 embryos. This demonstrates that here presented, chip protocol can be used to generate epigenomic profiles from limited amounts of various embryonic tissues.
By using this protocol to generate the histone modification profiles in an embryonic tissue, it is important to remember that signals will come from all different cell types present in such tissue. Following this protocol and using more starting biological material, it should be possible to investigate the binding profiles of other proteins involved in transcriptional regulation, such as, transcription factors, co-activators or RNA polymerase 2 sub units. Once established, these protocols will enable researchers in the field of developmental biology or medical genetics to improve the functional alteration of vertebrate genomes and to gain important insights into the molecular basis of myogenesis, evolution and human disease.
