To analyze gene expression changes caused by RML induced neurodegeneration in a cell-type specific manner the RiboTag method was used. At first, cell-type-specific isolation of ribosome associated mRNA from Rtag/Cre mice by RiboTag IP had to be established in our laboratory and at the beginning it was done as described in the original publication (Sanz et al. 2009). However, the achieved cell-type-specific mRNA yields and cell type specificity compared to input total RNA were not satisfactory. Therefore, we tested several changes to the original protocol including incubation times, buffer ingredients, reaction volumes, order of reaction steps and anti-HA antibody isolates or clones, testing each parameter one at a time. To verify the impact of the changed parameters yields of cell-type-specific immunoprecipitated mRNA (Agilent 2100 Bioanalyzer and Qubit Fluorometer) and the cell type specificity (RT followed by qPCR; enrichment or depletion of cell type marker genes) were analyzed. Western blot was used to analyze the disposition of the HA tagged ribosomes during the IP, for example whether the ribosomes were bound and remained coupled to the beads during the procedure or dispersed in the supernatant or washing steps. Western blot was also informative about input volumes and amount of magnetic beads by checking abundance of tagged ribosomes bound to beads versus remaining in the supernatant. Dot blot was used to test different antibodies, their affinity to the magnetic beads and the needed antibody concentration. At the end, an improved RiboTag IP protocol was developed and thereafter IPs were done as described in the material and method section for this study (Section 3.7).
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One important example is we discontinued the use of heparin in the brain homogenate. Although, the addition of heparin in the polysome buffer leads to a higher yield of immunoprecipitated mRNA, these mRNAs are less cell-type-specific. The enrichment or depletion of marker genes was more significant compared to preparations made with heparin (data not shown).
In detail, additional to the exclusion of heparin in the brain homogenate changes to the original protocol are as follows. Instead of 200U/ml RNase inhibitor only 100U/ml was used. The reduction of RNase inhibitor to this concentration did not lead to any difference of yields and cell type specificity (data not shown).
We also tested whether the order in which antibodies and beads are added to the supernatant affects the IPs (direct vs. indirect IP method). In the direct method from the original protocol antibody-coupled beads are incubated with the brain homogenate. In the indirect method brain homogenate is first incubated with the antibody and this mixture is then added to the magnetic beads. Because the indirect method is prone to background contamination since the magnetic beads are not saturated with antibody and unspecific binding of ingredients from the brain homogenate supernatant to the beads can occur, brain homogenates were pre-cleared in advance with fresh, non-reusable magnetic beads. Elimination of the pre-clearing step within the indirect IP method led to contaminations in the mRNA and therefore to less cell-type specificity (data not shown). Changing the IP from the direct to the indirect method with addition of a pre-clearing step of the brain homogenate resulted in an increase of immunoprecipitated mRNA with constant cell type specificity (data not shown).
We also tested different anti-HA antibodies for our RiboTag IP. In our experiments anti-HA 12CA5 worked the best. Other tested antibodies led to less yields of immunoprecipitated mRNA even with high concentrations or did not even stay coupled to the beads (data not shown). The ideal antibody concentration was determined by a dot blot assay. The amount of anti-HA 12CA5 antibody bound to the beads and the amount of antibody left in the supernatant was analyzed with different antibody concentrations. The antibody concentration in which the beads were saturated with antibody but no or little antibody was left in the supernatant was used. This determined concentration for the anti-HA 12CA5 antibody was 10µl / 200µl supernatant (data not shown).
Furthermore, different IP reaction volumes were tested. In our experiment half of the input volume compared to the original protocol for RiboTag IP was used, so 200µl instead of 400µl. With less input we proportionately harvested more immunoprecipitated mRNA, especially for very abundant cell types (data not
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shown). We assume that this effect is caused by oversaturated magnetic beads and RNA isolation columns. If higher input volumes were used we harvested more cell-type-specific mRNA when the material was split on two RNA isolation columns than loading all material on one column (data not shown).
In addition, different incubation times were given a trial. Incubation times were tried to be done as short as possible to lower the risk of RNA degradation without any decrease in mRNA yields or less significant cell type specificity. The final developed procedure includes pre-clearing of supernatant for 30min, incubation of pre-cleared supernatant with antibody for 45min and incubation of all components together for 1-2h. To avoid additional risk of RNA degradation most steps of the IP protocol were performed at 4°C. Longer incubation times or extending the IP overnight like described in the original protocol had no advantages in immunoprecipitated mRNA yields or cell type specificity (data not shown).
In summary, our improved RiboTag IP protocol differs from the original in six ways: elimination of heparin, concentration of RNase inhibitor, change to indirect IP method with pre-clearing, use of anti-HA 12CA5 antibody, change of IP reaction volumes and IP reaction incubation times.
4.3.2 RNA-sequencing sample generation
After the selection of mouse genetic background, disease time points for planned analysis and improvement of the RiboTag IP, needed Rtag/Cre mice were bred to isolate actively translated mRNA from specific cell types of the brain to analyze effects on gene expression caused by intracranial RML injection (Sanz et al. 2009). To study the response to RML induced neurodegeneration, mice were intracranially injected with brain homogenate from either normal or prion infected (RML) mice. Homogenates were injected into the right brain hemisphere through the bregmatic suture (Figure 4.3.2.1C). Mice in a 129S4 genetic background expressing the epitope-tagged ribosomes in astrocytes (Astro, Cre driver: Cnx43) or subsets of neurons, including glutamatergic (Glut, Cre driver: Vglut2), GABAergic (GABA, Cre driver: Gad2), parvalbumin (PV, Cre driver: Pvalb) or somatostatin neurons (SST, Cre driver: SST) were used to study a wide range of cell types (Figure 4.3.2.1B). Mice were sacrificed after 10 or 18 wpi.
To harvest cell-type-specific mRNA, dissected, frozen mouse brain tissue was homogenized followed by an immunoprecipitation and RNA isolation. Cell-type-specific mRNA and total mRNA from brain homogenate IP input were