This protocol aims to simplify the preparation of low input, small RNA sequencing libraries from early embryos to accurately profile the microRNAs that regulate embryonic development. The advantage of this technique is that it avoids the pooling of low input samples and can easily be applied to both non-sorted and sorted cells. We also demonstrate careful embryo staging to avoid variants between replicates.
This method can be applied to temporal, genetic, and developmental studies or other applications where input RNA concentrations are low. In the future, this method could potentially be used to diagnose developmental disorders in which microRNAs are dysregulated. The most technically challenging part of this protocol is the embryo dissection.
Because each embryo must be dissected, stage, image, and then dissociated quickly to ensure cell viability. To dissect the embryos, rinse the uterus of a pregnant female mouse with PBS and place it in a sterile plastic 10 centimeter dish. Use microdissection scissors to separate the decidua containing region and peel off the uterine muscle to expose all decidua.
To dissect the embryos, rinse the uterus of a pregnant female mouse with PBS and place it in a sterile plastic 10 centimeter dish. Use microdissection scissors to separate the decidua containing region. Move all embryos into a new dish with clean PBS and take an image of the entire litter.
Then image each embryo individually, and count the number of somites, keeping the magnification and exposure constant between experiments. For embryos older than E8.0, decapitate just above the otic placode and transfer the head to a clean well of a 48-well plate. Add 250 microliters of papain to the well and pipette up and down gently.
Use the microscope to check for clumps and continue pipetting up and down until a single cell suspension is achieved. Then add 250 microliters of FBS to the cells and filter 500 microliters of the suspension through a 35 micrometer nylon mesh filter. Move the filtrate to a new 1.5 milliliter tube and spin it down at 200 x g for five minutes.
Transfer the supernatant to a new tube, and resuspend the cell pellet in 300 microliters of PBS with BSA. When ready to sort filter the cells again through a filter cap tube and add DAPI to stain live cells. Use a cell sorter with a 70 micrometer nozzle to sort each sample into 500 microliters of RNA extraction lysis solution, then mix and store the sample at negative 80 degrees Celsius.
Add five microliters of 6X loading dye to each sample and mix by pipetting. Load the samples onto a 6%TBE polyacrylamide gel starting with the latter in the first well and leaving one lane between each sample. Run the gel at 150 volts for 30 to 40 minutes in 0.5X TBE running buffer.
When the gel has finished running, carefully remove it from the glass plates and place it in the tray from its original packaging, noting the orientation. Remove the running buffer and stain the gel for 15 minutes. And then image it on a UV trans illuminator.
Identify the band at 150 base pairs and use a clean razor blade to excise it. Making sure to avoid the adapter dimer product at 130 base pairs. Put the gel slices into microcentrifuge tubes and spin them down at the maximum speed for 30 seconds.
Next, use a P200 tip to crush the gel slice into the smallest bits possible and eject the tip into the tube. Add 300 microliters of elution buffer to each tube, washing the gel bits from the side. Reattach the pipette tip and wash it off before removing it from the tube.
Incubate the samples overnight at 25 degrees Celsius with 1, 000 RPM shaking. On the next day, spin them down at maximum speed for 10 minutes and transfer the supernatant to an RNase-free 96-well plate. Add 50 microliters of cleanup beads to each sample and pipette to mix.
Then add 350 microliters of isopropanol, mix the sample and incubate the plate at room temperature for 10 minutes while rocking. After the incubation, pull spin the plate and magnetize it for two minutes. Discard the supernatant and wash the beads with 950 microliters of 80%ethanol for 30 seconds.
Dry the sample for three minutes, then remove all residual liquid at the bottom of the well. Remove the plate from the magnetic stand. And resuspend the beads in 13 microliters of the resuspension buffer.
Incubate the plate for two minutes, then transfer it back to the magnetic stand for three minutes. Transfer 12 microliters of the supernatant to a clean tube and store it overnight at four degrees Celsius or long-term at negative 20 degrees Celsius. Embryos at E7.5, E8.5, and E9.5 were harvested.
And somite staged to resolve the six hour time intervals. When principle component analysis was used to group samples based on similarity, it was found that samples cluster by age. Premigratory and migratory neural crest cells were labeled that E8.5 with Wnt1-Cre and an E9.5 with Sox10-Cre.
A comparison of the two CRE drivers confirms that they mark different populations in early mouse embryos. RNA was quantified with a spectrophotometer and capillary electrophoresis. While the spectrophotometer trace revealed that the RNA contains no contaminating proteins and a high cell concentration, it was not sensitive enough to detect changes in RNA concentration with age.
The capillary electrophoresis trace can be used to estimate concentration and size of the RNA fragments. Peaks at 2, 000 and 5, 100 nucleotides are 18S and 28S ribosomal RNA, respectively. While the small RNA region is located at about 150 nucleotides.
Gel extraction can be used to isolate the small RNA sequencing libraries away from the adapter primers. Before excision, a multitude of product sizes are present in each sample. Capillary electrophoresis traces before and after size selection show the improvement in the purity of the 150 base pair library product after gel purification.
We have designed this protocol to split an RNA prep from a single sample into both small RNA sequencing libraries and bulk mRNA sequencing libraries. This will address questions surrounding not only microRNA expression, but also which mRNA targets may be regulated by the expressed microRNAs. This technique enabled us to obtain a comprehensive view of the most highly expressed microRNAs and the regulation of their targets in cranial neural crest cells between gastrulation and neural tube closure.
