Serial coronal brain sections at the level of the striatum (bregma 1 mm to 0.5 mm), were prepared using a cryostat (Leica) at –18°C and were mounted on Starfrost glass slides (Sigma Aldrich) for single cell isolation (8 μm thick sections) and for micro region isolation (20 μm thick sections) on PEN-membrane slides (Molecular Devices). Once the first section had been mounted, the slide was kept inside the cryochamber (-18°C) while the next section was cut. Eight to twelve sections were mounted in this way on each slide. Cutting and mounting was performed as quickly as possible to ensure that all sections adhered properly to the membrane. Two slides were prepared at a time and processed in parallel. After sectioning, the slides were stored on dry ice until dehydration and staining. Sections mounted on PEN slides (for micro region isolation) were stained prior to dehydration. Slides were fixed in 70% EtOH for 30 sec, rehydrated in H2O for 10 sec stained in 2% thionin staining solution (Sigma) washed two times in fresh H2O. For dehydration sections were subsequently fixed in 70% EtOH for 30 sec, followed by a dehydration series in 95% EtOH for 30 sec, two times ‘freshly’ pured 100% EtOH for one min. and two times Xylene for two min. After dehydration slides were air dried, transported and kept in a box with desiccant. Sections for single cell isolation were only subjected to dehydration procedure. We used only precooled RNAse free plasticware and solutions; The EYFP+ cells were microdissected from the motor and somatosensory cortex from 10 slides in total; we used the HS Transfer Caps (Molecular Devices) to capture between 100 and 300 neurons per cap, for a total sample size between 700 and 800 microdissected neurons. The corresponding CC-WM microregions were microdissected from 10 adjacent slides and were also pooled. For the microdissection, we used the Arcturus Veritas microdissection system with fluorescence package (Molecular Devices). The isolation of EYFP+ cells was done by identifying single bright fluorescent cells in the fluorescence mode. Cells were only collected if no adjacent nuclei were detected in close proximity. Successful cutting and collection steps were subsequently validated in bright-field and fluorescent mode on the quality control slot of the device. Microdissected samples were lysed in 100 μl of RNA lysis buffer (Qiagen, Hilden, Germany) byvigorous vortexing for 30 sec and stored at -80°C until further use. All procedures were done under RNAse-free conditions.
Growth protocol
For this study 1, 3, 6 and 12 month old male Cnp1-/-, Plp1-/Y and age-matched control mice (n = 3/genotype) were sacrificed by cervical dislocation. To reduce biological variation, all mice were processed between 9 and 10 h after light onset. Brains were removed from the skull and bisected along the midline using a razorblade. One half was frozen on dry ice, sealed with parafilm and stored at –80°C until further use and the other half was post immersion fixed in 4% paraformaldehyd (PFA) for 48 hours and paraffin embedded.
Extracted molecule
total RNA
Extraction protocol
‘RNeasy mini preps’ (Qiagen) were used to purify the total RNA from e.g. FACS-sorted microglia, glial precursor cells and oligodendroytes as well as for brain lysates. Total RNA was purified following the manufacturer’s instructions. In brief, cells or tissue were lysed in RLT buffer and kept at -20°C until further use. The samples were thawed quickly by incubating them at 37°C in a water bath and further homogenized by vigorous vortexing for 1 min. 100% EtOH was added to provide the appropriate binding conditions, and the homogenates were applied to RNeasy mini columns where the total RNA binds to the membrane and the contaminants are efficiently washed away. The RNAs were eluted from the column with 100 μl of RNase-free ddH2O.
Label
biotin
Label protocol
Total RNA of pooled single cells or micro-regions was resuspended with pretested T7- tagged dT21V oligonucleotides. Two-round T7-RNA polymerase-mediated linear amplification was performed according to optimized protocols for low-input RNA amounts (see Small Sample Target Labeling Assay Version II; Affymetrix). Biotinlabeled second-round aRNA was generated with a NTP-mix containing Biotin-11-CTP and Biotin-16-UTP (PerkinElmer, Boston, MA) (2 mM f.c.). Biotin-labeled amplified RNA (aRNA) size distribution and quantity was analyzed with the Agilent 2100 Bioanalyser using the RNA 6000 Nano LabChip kit (Agilent Technologies, Boeblingen, Germany). Samples with lower size compressed RNA products were discarded.
Hybridization protocol
At least 5 μg of labeled cRNA was fragmented by heating the sample to 95°C for 35 min in a volume of 20 μl containing 40 mM Tris acetate, pH 8.1, 100 mM KOAc, and 30 mM MgOAc. Fragmentation was checked by alkaline agarose electrophoresis. Hybridization, washing, staining, and scanning were performed under standard conditions as described by the manufacturer. Mouse430A 2.0 genechips were used that contain over 22,600 probe sets representing transcripts and variants from over 14,000 well-characterized mouse genes.
Microarray raw data (.cel files) were exported using Gene chip operating software (gcos) (Affymetrix). Normalization and higher-level analysis were done in R environment employing affy, simpleaffy, limma and several other packages downloaded from Bioconductor (detailed list see section 5.1.1.2). Data normalization, filtering and determination of significance cut-offs were performed following rigorous standards, optimized to achieve a minimum of false positive discoveries. Briefly, normalization was carried out using the Robust Multichip Average (RMA) model implemented in the R package Affy, using default settings.
