GSM8291588: CSS_VEH_VTA_DA_neuron_0009; Mus musculus; RNA-Seq
At 3 days after completion of CSS/CON, mice were deeply anaesthetized and then perfused with PBS (20 mL) at RT. The brain was removed and placed in a cryo-mould (E6032-ICS, Sigma) with embedding medium (Tissue-TEK OCT Compound). The cryo-mould was then placed on dry ice, wrapped in aluminium foil and a polythene bag and stored at -80°C. Frozen brains were processed using RNA- and RNAse-free conditions throughout. Using a cryostat set at -18°C, coronal sections that included the VTA at AP -2.9 to -3.3 mm were cut at 10 µm and mounted (3 sections/slide) on RNAse-free PET membrane slides (50102, Molecular Machines & Industries, MMI). Sections then underwent fixation and dehydration: 100% ETOH at RT for 20 s and xylene at RT for 20 s. Slides/sections were placed on their edge in a covered boy at RT for 10 min or until completely dried, and then in a capped 50 ml Falcon tube for storage at -80°C for 3 days maximum. Tissue samples that were EGFP+ were collected from these coronal sections using a laser capture microdissection (LCM) system (CellCut, MMI). Fluorescence settings were optimized for visualization of EGFP+ tissue (channel FITC) or mScarlet-I+ tissue (channel TRITC). The membrane slide was positioned and using 4x magnification, VTA tissue areas that were EGFP+ were each encircled at Ø=35 µm using the MMI CellTools software. Selected EGFP+ areas that were also mScarlet-I+ were deselected. There were 20-30 EGFP+/m-Scarlet-I- samples per VTA hemisphere/section; these were encircled for both hemispheres for each of the 3 sections on the membrane slide. An MMI Universal UV laser (355 nm, 2 µJ, 4 kHz frequency, 500 pico-s pulse-duration) at 88% laser power was activated (velocity=51 µm/s, focus=2233 µm) and the designated tissue areas were collected on the adhesive cap of an MMI isolation tune (0.5 ml). The procedure was conducted with 3 membrane slides (7-9 sections) and isolation tubes per mouse, to yield a total of 500 EGFP+/mScarlet-I- tissue samples per mouse; this was with the exception of one CSS mouse in which the EGFP/mScarlet-I signals were weak (likely due to misplaced injection), and this mouse was excluded from the experiment. Following tissue collection, tissue lysis was conducted by adding QIAzol (100 µl) to the tube, triturating the tissue on the cap with 20 µl volumes and returning this volume to the tube; the tube was closed, inverted for 15 min at RT, and vortexed for 1 min, inverted for 5 min and centrifuged for 5 s. The tube was then sealed with Parafilm and frozen at -80°C until RNA extraction. Per mouse sample, lysate aliquots (3 x 100 µl per sample) were pooled to give a final lysis volume of 300 µL. Samples were transferred to 2 mL PhaseLock tubes (QuantaBio). A half volume of chloroform:isoamyl alcohol (24:1 v:v) was added before shaking, 3 min RT incubation and centrifugation at 4°C. The aqueous phase was then transferred to a 1.5mL Eppendorf tube and mixed with a 1.5 volume of isopropanol (Sigma). After thorough pipette mixing, the isopropanol mixture was applied to a RNeasy MinElute spin column and total RNA was extracted using the miRNeasy Micro Kit (Qiagen) with a DNase treatment. Samples were eluted in 14 µL nuclease-free water. RNA samples were assessed both quantitatively and qualitatively using the High Sensitivity Total RNA 15nt Analysis DNF-472 Kit on a 48-channel Fragment Analyzer (Agilent). Total RNA yield was 1.14 ± 0.20 ng; RNA integrity could often not be computed due to low input. Up to 1.4 ng of total RNA was used for cDNA synthesis, conducted with the SMART-Seq® v4 Ultra Low Input RNA kit (Takara Bio); 12 amplification cycles were conducted. After clean-up, up to 10 ng of cDNA was used to generate the final sequencing libraries with the tagmentation-based DNA Prep Kit (#20018705) and the IDT® DNA/RNA UD Indexes Set A (#20026121), both Illumina®. The index PCR was performed with 9 cycles, while the final library was eluted in 30 µL EB Buffer. Low input mRNA libraries were then quantified using the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) on a Synergy HTX (BioTek). Library molarity averaged 42 nM. Libraries were also assessed for size distribution and adapter dimer presence (10, Rd3: 10, Rd4: 101), reaching an average depth of 26 million Pass-Filter reads per sample (14.2% CV).