TRAP-rc, Translating Ribosome Affinity Purification from Rare Cell Populations of Drosophila Embryos

The overall goal of this procedure is to isolate engaged in translation RNA in a cell typed specific manner in drosophila embryos. This is accomplished by first collecting embryos from a transgenic fly line, allowing specific expression of A GFP tagged ribosomal subunit in the targeted cell type, in this case, the slouch muscle cells. The second step is to generate a lysate to obtain a polysome preparation containing a mixture of tagged and untagged ribosomes.

Next, the protein concentration in the lysate is estimated and affinity purification is performed with beads coupled to GFP antibodies to capture ribosome RNA complexes from the cell type of interest. The final step is dedicated to triol, RNA, extraction and purification. Ultimately, quality and specificity assessments are performed with a bioanalyzer and R-T-Q-P-C-R respectively.

RNA can then be used for global quantitative analysis by RNA sequencing or microarrays. The main advantage of this technique of our existing method, like VX sorting, is that it does not require a labo step of cell dissociation. It can be performed on the bench.

It's fast and allows direct identification of translated RNA in very small population of cells, which is a measure benefit for the understanding of the acquisition of cell properties. Even if this method can provide insight into myogenesis in Josephine embryo, it can be also applied to other tissue or modern organism, such as plant, zebra, fish, or mouse, and could also help following impact of treatment on protein synthesis in case of pathology. Demonstrating the procedure will be Benjamin Berta, a PhD student from my laboratory After engineering, both transgenic lines according to the instructions in the written portion of the protocol and crossing to create the trap double cross transgenic line, amplify the line by first preparing eight large cylindrical population cages containing around 40 grams of young flies per cage, which corresponds to around 180, 000 flies per cage.

Prepare 11 centimeter diameter Petri dishes containing a mixture of solidified agar and grape juice with one third of the surface covered with freshly made yeast paste, and allow the flies to lay eggs on these plates for one hour. Repeat this process on two further sets of fresh grape juice plates to allow the flies to fully empty their ucts. The desired double transgenic embryos from the cross will be laid on the fourth plate.

Remove the plates containing the desired embryos from the cages and allow them to incubate to the desired developmental stage at room temperature. A 13 hour incubation is used here. Then resuspend the plate content with around 50 milliliters of water using a brush.

Separate the embryos from the dead flies by passing the liquid through three sieves of 700, 355 and 112 micron diameters. Rinse collected embryos in the smallest diameter sieve with the ionized water. Coate the embryos in 4.5%bleach in the ionized water for two minutes, and then rinse thoroughly with the ionized water for 30 to 60 seconds.

Incubate the embryos in PBS 0.01%T between 20 and 100 micrograms per milliliter. Cyclo heide for five to 10 minutes at room temperature with agitation. After drying the embryos on cellulose absorbent sheets, transfer them to micro centrifuge tubes, then weigh the tubes and flash, freeze the embryos by immersion in liquid nitrogen.

In this stage, the dried embryos can be stored for several months at minus 80 degrees Celsius. Trend transfer 1.5 grams of the dried embryos to pre chilled 15 milliliter tubes, containing four milliliters of polysome extraction, buffer and homogenized. Using a sterile 10 milliliter serological pipette.

Then spread the homogenized embryos into two pre chill, two milliliter tubes and grind the embryos in a multi-directional fast speed bead grinder. Set to run twice for 10 seconds at 5, 000 RPM with a 15 second pause between each cycle. After 10 minutes of centrifugation at 2000 G, transfer the lysate into a fresh pre chilled micro centrifuge tube on ice.

Add 10%non P 40 to the S supernatant to a final concentration of 0.1%and mix gently by inverting the tube. Add 300 micromolar of DHPC to a final concentration of 30 millimolar and mixed gently by inverting the tube. Incubate the mixture on ice for five minutes, mixing by inversion several times during incubation.

Following the incubation in ice. Prepare the post mitochondrial supinate by centrifugation at four degrees Celsius for 10 minutes at 20, 000 G.After measuring the protein concentration by the Bradford Method and obtaining a protein concentration between 60 to 80 milligrams per milliliters, transfer the SNAT to a fresh pre chilled micro centrifuge tube and proceed immediately to the pre absorption step. Resuspend the magnetic beads with gentle agitation and then transfer 30 microliters of beads for each milliliter of lysate into a micro centrifuge tube.

Collect the beads on the magnet, pipette off the snat and resuspend the beads and 500 microliters of polysome extraction buffer. Next, collect the beads on the magnet. Again, add embryonic, lysate and incubate for one hour at four degrees Celsius on a rotator.

Remove the beads and place the supine agent on ice until the immuno purification step. Reserving 100 microliters of the supinate as the global RNA sample for comparison with the sample collected after immuno purification to immuno purify the sample, add one milliliter of pre absorbed lysate to an RNA free tube containing 90 microliters of blocked beads coupled to GFP antibody. Incubate the sample at four degrees Celsius for two hours or overnight with gentle end over end mixing in a tube rotator following the incubation.

Wash the beads by first spinning down the remaining beads in a mini centrifuge. Then collect on a magnet resuspend in 500 microliters of wash, buffer and incubate by and over. End mixing 10 minutes in the cold room and again, collect the beads on the magnet.

Transfer the beads to a fresh pre shield R RNAs free tube and wash twice more. Finally, after collecting the beads on the magnet, proceed to RNA extraction by adding one milliliter of TRIOL to the beads and performing extraction according to the manufacturer's instructions. Follow this by RNA cleanup, using an RNEZ micro kit according to the manufacturer's instructions.

To test the specificity of the trapped RNA perform reverse transcription on three nanograms of immuno purified and input RNA according to standard procedures. Then use the CDNA obtained for QPCR with gene specific primer sets. This confocal image of a stage 16 embryo shows RPL 10 A-E-G-F-P expression, specifically in the six slouch muscle cells in each HEMI segment, colocalization of RPL 10 A-E-G-F-P with the general muscle marker beta three tubulin is observed.

Trapped RNA was run on a bioanalyzer for quality control purposes. Note the perfect integrity of one eight s and two eight s ribosomal RNAs. This figure shows the results of an RT QPCR analysis on three biological replicates of stage 16 embryos, and demonstrates the high specificity of trap isolated mRNA.

FO change was calculated compared to input and normalized against the RPL 32 gene meth. Two transcripts present in all muscle lineages are 2.3 fold enriched compared to input, whereas more restricted slouch transcripts are 5.6 fold enriched neural cells expressing prospero and socks and genes are depleted 2.2 fold and 5.5 fold respectively After its development. This technique paved the way for researchers, particularly in the field of developmental biology, to explore self aid commitment and acquisition of cell properties during differentiation of doof embryos.