title: GSM6797613: run1_mouse14-intBA_NAc; Mus musculus; RNA-Seq
library_construction_protocol: At 29 days post-surgery, mice were deeply anaesthetized and then perfused with PBS at RT for 4 min to rinse blood from the brain. The brain was removed and placed in a cryo-mold (E6032-1CS, Sigma-Aldrich) with an embedding medium (Tissue-TEK OCT Compound). The cryo-mold was then placed on powdered dry ice for 10 min, wrapped in aluminium foil, placed in a plastic bag and stored at -80°C, prior to laser capture microdissection. Frozen brains were processed using RNA- and RNAse-free conditions throughout. Using a cryostat set at -17 to -20°C, coronal sections that included the NAc at +1.6 to +0.5 were cut at 40 µm, mounted on microscope slides (SuperFrost, Epredia) and processed to estimate the CTB injection site. Then, coronal sections that included the BA at -1.6 to -2.0 and -2.3 to -2.6 were cut at 10 µm and mounted (3 sections per slide) on RNAse-free PET membrane slides (50102, Molecular Machines & Industries, MMI). Sections then underwent fixation and dehydration using the following steps: 70% ETOH at -20°C for 120 s, dipping in/out ddH20 ice-cold for 60 s, dipping in/out 70% ETOH ice-cold for 60 s, 95% ETOH ice-cold for 45 s, 100% ETOH at RT for 120 s, 3x washing in xylene at RT, xylene at RT for 120 s. Slides/sections were placed on their edge in a covered dark box at RT for 10 min or until completely dried, and then in a capped 50 ml Falcon tube for either immediate cell collection or stored at -80°C for 7 days maximum. Tissue labelled with CTB-555 was collected from coronal sections at bregma AP -1.6 to -2.0 and -2.3 to -2.6 using a laser capture microdissection system (CellCut, MMI). Fluorescence settings were optimized for visualization of CTB-555+ tissue (channel TRITC). The membrane slide was positioned and using 40x magnification, BA tissue areas that were CTB-555+ were each encircled at Ø = 35 µm using the MMI CellTools software. There were 40-60 such areas per BA hemisphere and these were encircled for both hemispheres for each of the 3 sections on the membrane slide. A MMI Universal UV laser (355 nm, 2 µJ, 4 kHz frequency with 500 pico-s Pulse-Duration) at 40-50% laser power was activated (velocity = 54 µm/sec, focus = 27 µm) and the designated tissue areas were cut out and collected on the adhesive cap of an MMI Isolation tube (0.5 ml). The procedure was conducted with 2 membrane slides (6 sections) and isolation tubes per BA region and mouse,  giving a total of 500-510 CTB-555+ tissue samples per BA region and mouse. Following tissue collection, tissue lysis was conducted by adding QIAzol® (100 µl/tube) 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 sec. The tube was then sealed with Parafilm and frozen at -80°C until RNA isolation. Lysate aliquots (2-3 x 100 µl per sample) from the same BA sample were pooled, and, if required, QIAzol was added to give a final lysis volume of 300 µL. Samples were transferred to 2mL PhaseLock tubes (QuantaBio). A half volume of chloroform:isoamyl alcohol (24:1 v:v) was added prior to 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 an 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 1.5 ng of total RNA was used for cDNA synthesis, conducted with the SMART-Seq® v4 Ultra Low Input RNA kit (Takara Bio). Depending on the input amount, 12-15 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 or 10 cycles depending on cDNA amount, 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 at 21 nM. Libraries were also assessed for size distribution and adapter dimer presence (<0.5%) by the High Sensitivity NGS Fragment DNF-474 Kit on a 48-channel Fragment Analyzer (Agilent). All sequencing libraries were then normalized on the MicroLab STAR (Hamilton), pooled and spiked-in with PhiX Control v3 (Illumina). The library pools were subsequently clustered on an SP Flow Cell and sequenced on a NovaSeq 6000 Sequencing System (Illumina) with dual index, paired-end reads at 2 x 100 bp length (Read parameters: Rd1: 101, Rd2: 10, Rd3: 10, Rd4: 101), reaching an average depth of 26 million Pass-Filter reads per sample (18.2% CV).