The overall goal of the following experiment is to determine DNA regions that are associated with a specific non-coding RNA using QPCR or high throughput sequencing. This is achieved by cross-linking cells to trap native D-N-A-R-N-A interactions in vivo. As a second step cells are lysed and subjected to sonication to solubilize the cell lysate and sheer chromatin into small fragments.
Next biotinylated antisense oligos are added to the cell lysate in order to specifically enrich for the R-N-A-D-N-A complexes of interest results are obtained that elucidate the identity of non-coding RNA bound genomic sites based on QPCR or high throughput sequencing. The main advantage of this technique over existing methods such as R-N-A-D-N-A FISH, is that chirp allows genome wide maps of non-coding RNA binding sites at near base pair resolutions. This method can help us answer key questions in the non-coding RNA field, such as how the rocks, RNAs, and male flies achieve dosage compensation.
The implications of this technique, the extent towards therapies of non-coding NA related diseases because it allows to better understand how non-coding RNA regulate chromatin states and gene expression. For this chromatin isolation by RNA purification or chirp experiment, 40 million cells will be harvested after centrifugation, decant the PBS and remove the remaining liquid by carefully aspirating at an angle dislodged the cell pellet by tapping the bottom of the falcon tube. Add freshly prepared 1%glutaraldehyde, starting with a small volume and resuspend the cell pellet top up to the full volume and then invert to mix cross-link for 10 minutes at room temperature on an end-to-end shaker or rotator quench the cross-linking reaction with one 10th volume of 1.25 molar glycine and leave on a shaker at room temperature for five minutes, the cells will turn bright orange.
Next spin at 2000 RCF for five minutes, decant and aspirate the supernatant and wash the pellet with 20 milliliters of chilled PBS spin again, aspirate the supernatant and resuspend the washed cross-linked palate with chilled PBS. Transfer each milliliter of cells to an einor tube and spin at 2000 RCF for three minutes at four degrees celsius. After centrifugation, use a pipette tip to carefully remove as much PBS as possible.
Flash, freeze all the cell pellets in liquid nitrogen and store at minus 80 degrees Celsius indefinitely. To prepare cell lysate for chirp, thaw the frozen cross-linked cell pellets at room temperature. Tap hard to dislodge and mix the cell pellet.
Spin down the pellets and use a sharp 10 microliter pipette tip to remove any remaining PBS on an electronic balance that is accurate to one milligram. Tear the mass of an empty einor tube. Weigh each pellet and record its weight.
Add 10 x volume of lysis buffer supplemented with fresh protease inhibitor PMSF and super ace in to each tube and resuspend the pellet. The cell lysate must be ated immediately after its preparation. Transfer no more than 1.5 milliliters of lysate into each 15 milliliter falcon tube and insert the sonication probes screwing them on tightly.
Next place the tubes in a bio rupture, sonicate at four degrees Celsius at the highest setting with 30 seconds on 45 seconds off pulse intervals. Check lysate every 30 minutes. Continue sonicate until the cell lysate is no longer turbid.
This may take from 30 minutes to four hours depending on the number of tubes, the sample volume, the bath temperature, and the period of sonication time. When the lysate turns clear, transfer five microliters to a fresh einor tube. Add DNA protease K buffer and protease K vortex to mix and spin down briefly incubate for 45 minutes at 50 degrees Celsius after extracting DNA with a PCR purification kit.
Check DNA size on a 1%aros gel. The bulk of the DNA smear should be 100 to 500 base pairs indicating complete sonication centrifuge. One milliliter aliquots of sonicated samples at 16, 100 RCF for 10 minutes at four degrees Celsius, transfer the supernatant into fresh einor tubes and flash freeze in liquid nitrogen to begin the procedure for chirp thaw tubes of chromatin at room temperature for a typical chirp sample.
Using one milliliter of lysate, remove 10 microliters for RNA input and 10 microliters for DNA input and place in einor tubes. Keep on ice till further. Use transfer one milliliter of each chromatin sample to a 15 milliliter falcon tube.
Add two milliliters hybridization buffer to each tube. Next, thaw the probes at room temperature. These biotinylated tiling oligos are labeled according to their positions along the RNA and separated into two pools.
The even pool contains all probes with even numbers. And the odd pool contains probes with odd numbers. All experiments are performed using both pools which serve as internal controls for each other.
One of the most challenging aspects of the true protocol is that all hybridization and washing steps need to be performed at 37 degrees. To ensure consistent experimental temperature, perform all steps in or near a habitization oven, Add the appropriate volume of probes to specific tubes. Mix well incubate at 37 degrees for four hours with shaking 20 minutes before hybridization is complete.
Prepare the C one magnetic beads. Use 100 microliters per 100 pico mole of probes. Wash with one milliliter unsu supplemented lysis buffer three times using the Dyna mag two strip magnet to separate beads from buffer.
After the final wash, resus suspend beads in the original volume of lysis buffer and supplement with superin fresh PMSF and protease inhibitor. When the four hour hybridization reaction is complete, add 100 microliters of beads to each tube mix. Well incubate at 37 degrees Celsius for 30 minutes with shaking wash beads with one milliliter of wash buffer prewarm to 37 degrees Celsius on the first wash.
Use a dyna mag 15 magnetic strip to separate beads, decant and resuspend in one milliliter wash buffer, transfer to a 1.5 milliliter EOR tube. Incubate at 37 degrees Celsius with shaking for five minutes on subsequent washes, spin down each tube in a mini centrifuge and set sample on a dyna mag two magnetic strip for one minute decant sample. Wipe any drips with a Kim wipe and resuspend in one milliliter.
Wash buffer, incubate at 37 degrees Celsius with shaking for five minutes. Repeat for a total of five washes at the last wash Resus. Suspend the beads well remove 100 microliters and set aside for RNA isolation reserve 900 microliters for the DNA fraction.
Place all tubes on the dyna mag tube magnetic strip and remove the wash buffer. After a brief spin, place the tube again on the magnet strip with a sharp 10 microliter pipette tip. Remove the remaining wash buffer completely from each tube.
RNA and DNA fractions are subsequently isolated from the chirp samples for quantitation and sequencing. This figure shows enrichment of human telomerase, RNA or Turk from HELOC cells over GA dh. An abundant cellular RNA that serves as a negative control.
The majority of Turk RNA present in the cell, about 88%was pulled down by performing chirp, whereas only 0.46%of GA DH RNA was retrieved demonstrating an enrichment factor of approximately 200 fold Non-specific probes such as probes targeting LAC ZRNA, which is not usually expressed in mammalian cells, can be used as additional negative controls. DNA regions expected to bind the target. Long non-coding RNA are typically enriched over negative regions when measured by QPCR.
This figure shows QPCR validation of four hot air bound sites in primary human foreskin fibroblasts determined by performing chirp SSE in the same cell line while Turk and GA DH DNA sites serve as negative control regions. Both even and odd probe sets yielded comparable enrichment of expected hot air bound sites over negative regions. A hallmark of true long non-coding RNA binding sites.
A global map of long non-coding RNA binding sites can be obtained by high throughput sequencing of chirp enriched DNA as illustrated by this data from the trla long non-coating RNA Rocks two, which is known to interact with X chromosome in a manner that is required for dosage compensation. Both even and odd samples were sequenced separately and their unique noises eliminated to produce a track of overlapping signals where each peak indicates a strong sight of rocks two binding While attempting this procedure. It's important to exercise correct RNAs free technique Following this procedure.
Other methods such as protein do blotting can be performed in order to answer additional questions such as what proteins are interacting with the non-clinical DRNA of interest After its development. CHI paved the way for researchers in the RNA biology field to map the RN aact in culture cells and fixed tissues.
