GSM7018822: SCHW00151; Mus musculus; RNA-Seq
Fresh brain tissue was placed in ice-cold aCSF (2.5 mM KCl, 7 mM MgCl2, 0.5 mM CaCl2, 1.3 mM NaH2PO4, 110 mM choline chloride, 25 mM NaHCO3, 1.3 mM sodium ascorbate, 20 mM glucose, 0.6 mM sodium pyruvate, bubbled in 95% O2 /5%CO2). 200 μm coronal slices that included the LC were collected. Slices were transferred to a 32°C holding chamber containing oxygenated aCSF, where they recovered for at least 1 hour. tdTomato-expressing LC neurons were manually extracted from the slice using glass pipettes pulled from borosilicate glass (World Precision Instruments) to create an ~50 µm tip. The ends of glass pipette tips containing picked cells were snapped into individual wells of a 96 well plate containing 4µl lysis buffer: 0.1µl RNase inhibitor (Thermo Scientific), 0.1µl ERCC RNA spike-in (Invitrogen), 0.02µl 10% Triton (Sigma), 1µl 10mM dNTP (Invitrogen), 0.1µl 100µM dT (IDT; 5'-AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN), and 2.68µl H2O. Samples underwent primer annealing (72℃ for 3min) before placing on ice. Next, 6µl reverse transcription reagent was added to each sample: 0.14µl H2O, 0.25µl RNase inhibitor, 2µl 5X First Strand Buffer (Clontech), 0.5µl 100mM DTT (Invitrogen), 2µl 5M Betaine (Sigma), 0.06µl 1M MgCl2 (Invitrogen), 0.1µl 100µM TSO (Qiagen; 5'-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G), 0.95µl SmartScribe enzyme (Clontech). Samples were incubated at 42℃ for 90min, 70℃ for 5min, then held at 4℃. DNA preamplification solution (15µl) was next added to each sample: 2.1375µl H2O, 12.5µl 2X Kapa HiFi Hot Start Ready Mix (Kapa Biosystems), 0.25µl 10µM IS_PCR Primer (IDT; 5'-AAGCAGTGGTATCAACGCAGAGT), 0.1125µl Lambda Exonuclease (NEB). Samples were incubated at 37℃ for 30min, 95℃ for 3min, 21 cycles of [98℃ for 20s, 67℃ for 15sec, 72℃ for 4min], 72℃ for 5min, and then held at 4℃. DNA cleanup, quantification, dilution, tagmentation, and barcoding were performed by the Hartwell Center at St. Jude Children's Research Hospital using standard methods. Smart-seq2