Research in our lab examines how chromatin associated proteins, histone modifications, and small RNA pathway factors, work together in the context of development and transgenerational epigenetic inheritance. The RNA inheritance assay, using a GFP reporter, has become a very powerful tool. This protocol describes how to standardize the preparation, scoring, and passaging of worms and the preparation of chip samples, which can help to generate reproducible results.
One of the experimental challenges when studying germline-expressed transgenes is isolating the effects in the animal's germline. We're currently using whole animals for chip, but in the future, we would like to develop assays to examine chromatin specifically in the germline. Our laboratory is interested in histone methylation and proteins that recognize these marks.
Going forward, we would like to investigate the interplay between these chromatin readers and small RNA pathways in the context of RNAi inheritance at transgenes and the regulation of endogenous sequences. Prepare animals for the RNAi inheritance assay by picking three or four gravid Caenorhabditis elegans adults under the 35 millimeter standard NGM plates, seeded with OP50-1 bacteria. After four days, wash the gravid adult worms off the plates into 1.5 milliliter tubes using 800 microliters of M9 buffer, supplemented with Triton X-100.
Repeat the wash and pull the worms into one tube. After centrifuging the worms at 1, 000 G for 1.5 to 2 minutes, aspirate the supernatant, leaving 100 microliters without disturbing the worm pellet. To each tube containing the worm pellet, add 650 microliters of double-distilled water.
To isolate the C.Elegans embryos from the collected population, add 250 microliters of the freshly-prepared bleaching solution and start a timer. Everyone one to two minutes, vortex the tubes thoroughly. After five minutes, monitor the degradation of adult worms under the stereoscope.
Continue vortexing until the worms are completely dissolved and only embryos remain. This generally takes six to seven minutes. Immediately centrifuge the tubes at 1, 000 G for 1.5 minutes and aspirate the supernatant, leaving approximately 50 to 100 microliters of the supernatant with the embryos.
Add one milliliter of M9 buffer supplemented with Triton X-100 to the embryos and vortex. Centrifuge the suspension and repeat the wash two times. After the final wash, aspirate the supernatant and leave approximately 100 microliters in the tube.
Vortex the tubes with isolated embryos and pipette two microliters from each tube twice onto a labeled glass slide. Count the embryos using a tally counter to estimate the concentration of embryos per microliter. To start a semi-synchronized population growth, transfer approximately 250 embryos onto each RNAi NGM plate containing E.coli HT115 bacteria with GFP or control RNAi vectors.
Allow the liquid to absorb, incubate the P0 generation treated with RNAi for four days at 21 degrees Celsius. Begin preparing the agarose pads by placing a drop of melted 1%agarose under the vinyl record using a glass pasteur pipet. Orient a glass slide so that the lines on the record are horizontal or vertical and place the slide quickly onto the agarose drop for 30 seconds.
Remove the glass slide from the record and add approximately five microliters of freshly prepared five millimolar Levamisole under the stereoscope. Gently flick the tube containing worms and transfer five to 10 microliters onto the agarose pad to mount the worms. Estimate the number of worms as they are being added so that there are approximately 40 worms per replicate.
Line up the worms in rows using an eyelash pick, and place a glass cover slip onto the slide. Isolate the embryos from the remaining worms using the bleaching protocol and plate 250 embryos onto 35 millimeter NGM plates seeded with OP50-1 bacteria. Under the fluorescent stereoscope, use a GFP filter set and count and record the number of GFP positive and GFP negative worms on the slides for each replicate using a tally counter.
The manual scoring of the nuclear GFP signal in the germline for each generation showed that the silencing of the transgene was fully penetrant in the scored P0 and F1 worms treated with GFP RNAi. In the F2 generation, the proportion of the population exhibiting inheritance of GFP silencing was approximately 50%By the F5 generation, the majority of the population did not show the inheritance of silencing. By the F 10 generation, all the worms expressed GFP and no inheritance was detected.
Begin by plating approximately 3, 500 C.Elegans F1 generation embryos on 14 plates and allow them to grow to the young adult stage for three days at 21 degrees Celsius. Wash and collect the C.Elegans into 1.5 milliliter tubes using PBS/TX. Centrifuge at 1, 000 G for two minutes.
Aspirate and pull the worms from one strain into one tube. Repeat three washes with PBS/TX. Next, aspirate the supernatant, leaving one milliliter in the tube and invert to mix.
After counting the number of C.Elegans worms in three microliters three times, calculate the concentration of worms and transfer a volume corresponding to 3, 000 worms to a new tube for formaldehyde cross-linking. Begin by adding formaldehyde to a 1.8%final concentration to the tube containing the worms and rotating the tube at room temperature for six minutes. Then, immediately freeze the tubes in liquid nitrogen and store the samples at 80 degrees Celsius.
To proceed, thaw the formaldehyde crosslink sample in a room-temperature water bath for three minutes and rotate the samples for 16 minutes at room temperature. Add 1.25 molar glycine to a 125 millimolar final concentration and rotate for five minutes at room temperature. Following this step, keep the samples on ice or at four degrees and use ice-cold buffers until the magnetic bead dilution.
After centrifuging the sample at 1, 000 G for three minutes, wash it three times with one milliliter of PBS/TX. Then wash it two times with one milliliter of resuspension buffer. After the last wash, leave enough buffer to ensure the total volume is at least three times the pellet volume and a minimum of 100 microliters.
Begin by mixing and aliquoting 90 to 120 microliters of the formaldehyde crosslink C.Elegans samples into a polystyrene sonication tube. Add an equal volume of resuspension buffer containing 2x detergents. Sonicate in a water bath sonicator for seven minutes.
Gently mix the sample by pipetting and repeat the sonication for an additional seven minutes. Once done, transfer the sonicated lysate to a 1.5 milliliter tube. Add a half volume of resuspension buffer without detergents.
After centrifugation at 13, 000 G for 15 minutes at four degrees Celsius, divide the lysate supernatant into four parts. Store the input lysate part at 20 degrees Celsius in a 1.5 milliliter tube. Add 0.5 micrograms of Anti-H3K9 tri-methylation, anti-histone H3, or IgG, to the appropriate immunoprecipitation or IP sample.
Incubate the reaction at four degrees Celsius overnight with rotation. The following day, aliquot the nine microliters of Protein G coated magnetic beads per IP sample in a 1.5 milliliter tube. After two washes with one milliliter of FA 150, resuspend the magnetic beads in the FA 150 buffer.
Once done, add 7.5 microliters of the magnetic bead suspension to each IP antibody mix before incubating the tubes at four degrees Celsius for two hours with rotation. Next, wash the magnetic beads seven times using at least 200 microliters of each buffer solution. Incubate each wash at four degrees Celsius for five minutes with rotation before collecting the beads on a magnetic stand to aspirate the wash.
After aspirating the last wash of the buffer, resuspend the magnetic beads in 50 microliters of chromatin IP elution buffer, and transfer it to a 1.5 milliliter tube. After eluting the sample in a thermomixer, collect the beads on a magnetic stand and transfer the supernatant to a new tube. Repeat the elution with another 50 microliters of chromatin immunoprecipitation elucian buffer.
Once done, pool a total of 100 microliters of supernatant before proceeding with reverse cross-linking and DNA elution.
