title: GSM4172088: single neuron isolated from mouse striatum G1_1000701204-OGC16-sal_1_4ul_1__IL6985-728-510; Mus musculus; RNA-Seq
library_construction_protocol: Generation of D1-tdTom/D2-GFP, and D1-tomato BAC reporter mice was previously described(Gokce et al., 2016). All lines were maintained by backcrossing to C57Bl/6J. All other experiments were conducted on 5-7 week old male C57/Bl6 mice from Jackson Labs. Mice were weaned at ~21 days of age and housed in groups of 2-5 on a 12-hour light/dark cycle (lights on 0700-1900h), with free access to food. All procedures conformed to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved by the Stanford University. Parasagittal slices (350 μm) containing striatum were prepared using standard  procedures. Briefly, mice were anesthetized with isoflurane and decapitated,  brains were quickly removed and placed in ice-cold low-sodium/high-sucrose dissecting solution. Slices were cut by adhering the lateral surface of the brain to the stage of a Leica vibroslicer, and placed in ice-cold hibernate-A solution (Gibco, Themo Fisher). The striatum was dissected from each slice, and the tissue was dissociated using papain-based dissociation. Briefly, striatal tissue was incubated with frequent agitation at 30°C for 20 min 2mg/ml papain solution in Hibernate-A, followed by washing with hibernate-A including 1mg/ml Ovomucoid protease inhibitor (Sigma). The striatal slices were triturated briefly using fire polished glass Pasteur pipettes until a single-cell suspension was obtained. The suspension was cleaned from small debris by centrifuging for 3 min at 450g through a three-step density gradient of Percoll (Sigma), top layer containing small debris was discarded and remaining cells were pelleted for 5  min at 550g. The pellet was resuspended in 0.1 ml of hibernate A with DAPI (4',6-diamidino-2-phenylindole, 1:4000 dilution; Sigma) to label dead cells(Gokce et al., 2016). Striatal tissue was kept in ice-cold solution throughout the processing except during the 20 min dissociation with papain at 30°C. Administrative Panel on Laboratory Animal Care. The dissociated cell suspension from adult D1-tdTom/D2-GFP mice was passed through a 70-μm cell strainer (BD Biosciences). Viable cells (DAPI negative) were sorted by FACS (Aria II; BD Biosciences). For specific enrichment of genetically labeled cell types, fluorescent striatal cells were analyzed to determine optimal parameters and gating for FACS. Finally, test sorted cells were imaged on confocal microscope as described previously(Gokce et al., 2016). Cells were sorted into ice-cold hibernate-A and pelleted by centrifugation for 5 min at 300g. Single cells were sorted into 96 well plates filled with 4 μl lysis buffer composed of 0.05% Triton X-100 (Sigma) and, ERCC spike-ins (1:24000000 dilution), 2.5 μM oligo-dT, 2.5 mM dNTP and 2U/μl of recombinant RNase inhibitor (Clontech) then spun down and frozen at -80°C. Plates were thawed and libraries prepared as described below. 96-well plates with sorted single cells were incubated for 3min at 72°C and immediately placed on ice. To perform reverse transcription (RT) we added each well a mix of 0.59μl H2O, 0.5μl SMARTScribe™ Reverse Transcriptase (Clontech), 2μl 5x First Strand buffer, 0.25μl Recombinant RNase Inhibitor (Clontech), 2μl Betaine (5M Sigma), 0.5μl DTT (100mM) 0.06μl MgCL2 (1M Sigma), 0.1μl Template-switching oligos (TSO) (100μM AAGCAGTGGTATCAACGCAGAGTACrGrG+G). Next reaction RT reaction mixes were incubated at 42°C for 90 min followed by 70°C for 5 min. Pre amplification of cDNA done by adding 12.5μl KAPA HiFi Hotstart 2x (KAPA Biosystems), 2.138μl H2O, 0.25μl ISPCR primers (10 μM, 5′-AAGCAGTGGTATCAACGCAGAGT-3), 0.1125μl Lambda Exonuclease under the following conditions: 37°C for 30 min, 95°C for 3 min, 21 cycles of (98°C for 20 sec, 67°C for 15 sec, 72°C for 4 min), final extension at 72°C for 5 min. Cells were then cleaned using 2 rounds of AMPure bead (Beckman-Coulter) cleanup at a 0.8:1 ratio of beads to PCR product. The cleaned cDNA was then tagmented using the Nextera XT DNA kit and following the included instructions, using 11 cycles of PCR to amplify the tagmented cDNA (Illumina). Tagmented cDNA was then pooled and analyzed with a Bioanalyzer 2100 High Sensitivity DNA kit (Agilent) to assure that >80%  of the product was in the 200-1000bp range. This process was repeated separately for each animal. Libaries were sequenced using 2x75 reads base pairs (bp) paired-end on Illumina HiSeq 2000 or NextSeq to a target depth of 1x10e6 reads/sample.
attributes.tag: GEO Accession
attributes.value: GSM4172088