The overall goal of the following experiment is to identify genomic targets of KDM five a Jared one, a RBB two histone demethylase. This is achieved by preparation of cross-linked chromatin that is sonicated to produce DNA fragments of the appropriate size as a second step. RBB two bound DNA fragments are immuno precipitated with RBB two antibodies.
Next, the recovered genomic DNA is amplified by PCR for sequencing. Finally, Illumina sequenced short reads are uniquely aligned to the human genome, and significant peaks are identified and annotated to the closest genes. In order to identify rbbb two target genes results are obtained that show molecular functions associated with Rrb B two target genes based on genome-wide identification of Rbbb two enriched regions.
Hi, I'm Mike. She from the laboratory of Dr.Elizabeth Bencia in the Department of Biochemistry and Molecular Genetics at the University of Illinois in Chicago. And I'm ala from the laboratory of Biomedical genomics led by Dr.NGA at the BARCEL Biomedical Research Park University PO P fea.
And I'm Danielle Dal. And I'm William Richter. Both of us are also in the Benevolence Skill Lab.
Today we're gonna show you a procedure for chromatin, immunoprecipitation and sequencing. We use this procedure in our laboratory to study the binding site of his dermatologist. So let's get started.
To begin this procedure, grow approximately 10 to the eighth cells. For each immunoprecipitation or ip. Here, diffuse osteolytic lymphoma.
U 9 37 cells are used and induced for monocytic differentiation with TPA for 96 hours following cell growth at formaldehyde solution directly to the media to a final concentration of 1%Then swirl the flask briefly and allow them to sit at room temperature for 10 minutes. Then aspirate the media and rinse the cells with 15 milliliters of ice cold PBS. Repeat this wash once.
Next, add six milliliters of lysis buffer one to each of the flasks on ice. Then rock the flasks for 20 minutes at four degrees Celsius. Finally, harvest the cells using a cell scrapper and transfer them to 15 milliliter conical tubes.
At this point, the cells can be stored at minus 80 degrees Celsius. If the cells were frozen, haw them out. Once thawed, spin the cells down and discard the snat.
Then resuspend the cells in six milliliters of lysis buffer two, rock the tubes gently at room temperature for 10 minutes. Repeat centrifugation and resuspend the cells in 2.5 milliliters of lysis buffer three. Next, prepare the suspension for sonication by placing it in a beaker of icy water with the micro tip of a Branson 450 sonier set to between 50 and 60%amplitude.
Sonicate the solution for a 32nd constant burst and cool on ice. For one minute, repeat these pulses 10 to 15 times, then pipette the contents of the tube up and down and transfer them to a new tube. Continue to pulse and cool the solution.
An additional five times following sonication, add 10%Triton X 100 to one 10th of the solution volume. Then transfer the lysates to 1.5 milliliter centrifuge tubes, and spin the debris out in a micro centrifuge prior to chromatin, immunoprecipitation or chip. Save 50 microliters of the cell lysate as the input sample.
Then combined the cleared cell lysate with dyna beads bound to antibody that had been previously prepared as described in the written protocol. Rock the mixture in addition to the 50 microliter input sample at four degrees Celsius overnight. Wash the beads with one milliliter of wash buffer.
Then use a magnetic stand to collect the beads and remove the supernatant. Repeat this wash six to eight times. Perform a final wash with one milliliter of TE plus 50 millimolar sodium chloride.
Spin the beads in a micro centrifuge following centrifugation, aspirate any residual TE buffer. Then add 100 microliters of elu buffer to the samples. Immediately after.
Incubate the samples at 65 degrees Celsius for 10 to 15 minutes. Scratch the tubes against a 1.5 milliliter tube brack every two minutes to keep the beads in suspension. Collect the beads using centrifugation and the magnetic stand and transfer the SNAT to A PCR tube.
Then retrieve the previously saved input sample and add three volumes of the elution buffer. Finally, place the IP and input samples into the thermal cycler overnight at 65 degrees Celsius. To reverse the cross linkages the following day, add one volume of TE buffer to the samples.
Then add RNAs A to a final concentration of 0.2 micrograms per microliter. Incubate the samples in the thermal cycler for one to two hours at 37 degrees Celsius. After incubation, add proteinase K to a final concentration of 0.2 micrograms per microliter.
Then incubate samples in the thermal cycler at 55 degrees Celsius for two hours. Next, extract the samples once with one volume of phenol. Extract the samples a second time with one volume of phenyl chloroform isoamyl alcohol.
Finally extract the samples once more with one volume of chloroform isoamyl alcohol. Then add 30 micrograms of glycogen to each sample. Also, add sodium chloride to a final concentration of 0.2 molar and two volumes of ethanol to the samples.
Incubate them for 30 minutes at minus 80 degrees Celsius. After incubation, spin the samples and decant the snat. Wash the pellets with 500 microliters of 75%ethanol, then dry the pellets and resuspend them in 40 microliters of water.
To check the sizes of the DNA fragments produced by the SONICATION procedure, load five micrograms of the purified input sample in a 1.8%agro gel and run the gel. The sonication procedure should produce fragments in the range of 150 to 350 base pairs. However, if the fragments do not fall in this range, the sonication procedure should be adjusted by varying the number of bursts and the amplitude prior to amplification of genomic DNA.
Check the size, concentration, and enrichment of the samples as described in the written protocol. To begin amplification of genomic DNA, obtain 34 microliters of the IP sample and 200 nanograms of the input sample. Perform and repair atail addition and ligation of adapters to DNA fragments using an Illumina genomic DNA sample prep kit as described in the written protocol.
Purify the DNA using a mini lut PCR purification kit and then elute it in buffer EB that has been prewarm to 50 degrees Celsius. Use 23 microliters and 46 microliters for the IP and input DNA samples respectively. Make the PCR reaction using 23 microliters of DNA with adapters 25 microliters of fusion, DNA polymerase from the kit.
One microliter of 20 micromolar sole PCR one and one microliter of 20 micromolar. So PCR two run the PCR reaction as described in the written protocol following PCR amplification. Purify the DNA with the Qiagen mini loop PCR purification kit.
Prewarm 15 microliters of buffer EB to 50 degrees Celsius and dilute the DNA dilute 0.5 microliters of the sample one to four and perform an NanoDrop reading to purify the amplified products. Prepare a 1.8%AGROS gel of 50 milliliters using HPLC grade water add TAE into and bromide after the agros has melted. Then pour the gel, load the gel.
After adding four microliters of loading buffer to the input and IP samples, then run the gel at 120 volts for 45 minutes. Analyze the gel after it is run. The should reveal that fragments in the range between 150 and 350 base pairs are produced.
They represent genomic DNA fragments between 50 and 250 base pairs in length. And an adapter excise the region of gel containing the material in the 150 to 350 base pair range with a scalpel. Take care to avoid excising the adapter adapter band, which runs at about 120 base pairs.
Recover the DNA using a kayak quick gel extraction kit per manufacturer's instructions. Dry the sample down to precisely 11 microliters in a speed back without heat. Remove one microliter of the sample and measure the DNA concentration using a NanoDrop spectrophotometer.
Finally, identify enriched gene products using the Illumina genome analyzer and analyze the binding sites and target genes as described in the written protocol. Genomic coordinates of identified enriched RB binding protein two or RBP two binding regions are presented in a chromosome wise format. Occupancies of ensemble genes in the chromosomes can be visualized as black VARs.
Here chromosome 10 occupancies are shown rbp two binding regions are shown as vertical lines. The middle panel shows all rbp two isoform binding regions on chromosome 10, and the bottom panel shows rbp two large isoform binding regions. This information gives an overview of overlapping and specific occupancy of RBP two isoforms on chromosome 10.
Here a heat map is shown with false discovery rate or FDR corrected significantly enriched gene ontology or go molecular function categories among the genes bound by rbp. Two colors toward red indicate high statistical significance. Yellow indicates low statistical significance, and gray indicates no statistical significance.
Enrichment analysis shows the overlapping and isoform specific molecular functions of R BP two target genes. We have just shown you how to identify transcription factor binding sites During this procedure. It's important to produce DNA fragments of the appropriate size range and yield.
This can be achieved by following our guidelines during the SONICATION step. So that's it. Good luck with your experiments and thanks for watching.
