GSM1477079: SingleCellNo2824; Mus musculus; RNA-Seq
Mice were deeply anesthetized with ketamine/xylazine (80mg/kg; 10mg/kg). The brain was quickly dissected and transferred to ice-cold oxygenated cutting solution (87 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 75 mM sucrose, 20 mM glucose, 1 mM CaCl2, and 2 mM MgSO4) and kept in the same solution during sectioning on a vibratome (VT1200 S, Leica) in 300 µm thick slices. The area of interest was dissected from each slice, and the tissue was dissociated using the Papain dissociation system (Worthington) following the manufacturer's instructions. The cell suspension obtained was filtered with 20 µm filter (Partec) and kept in cold HBSS solution (SIGMA) with 0.2% BSA and 0.3% glucose. For FACS of EGFP cells, a BD FACSAria" III Cell Sorter B5/R3/V3 system was used. The cell suspension with 600-1000 cells/µL had C1 Suspension Reagent added (all 'C1' reagents were from Fluidigm, Inc.) in a ratio of 4 µL to every 7 µL cell suspension, and 11 µL of the cell suspension mix was immediately loaded on a C1 Single-Cell AutoPrep IFC microfluidic chip designed for 10- to 17-μm cells, and the chip was then processed on a Fluidigm C1 instrument using the 'mRNA Seq: Cell Load (1772x/1773x)' script (30 min at 4°C). Lysis mix, RT mix and PCR mix (described in Islam et al., Nat Methods. 2014 Feb;11(2):163-6) were added to the chip. The plate was placed in the Fluidigm C1 instrument and the 'mRNA Seq: RT + Amp (1772x/1773x)' script was executed, and included lysis, reverse transcription and 21 cycles of PCR. The amplified cDNA was harvested in a total of 13 μL C1 Harvesting Reagent. Amplified cDNA was simultaneously fragmented and barcoded by tagmentation using Tn5 DNA transposase to transfer adaptors to the target DNA as described (Ibid.). 100 µl Dynabeads MyOne Streptavidin C1 beads (Invitrogen) were washed in 2× BWT (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween-20) and resuspended in 2 ml 2× BWT. Twenty microliters of beads were added to each well and incubated at room temperature for 15 min. All fractions were pooled, the beads were immobilized and the supernatant removed. The beads were resuspended in 100 μL TNT (20 mM Tris, pH 7.5, 50 mM NaCl, 0.02% Tween), washed in 100 μL Qiagen Qiaquick PB, and twice in 100 μL TNT. The beads were resuspended in 100 μL restriction mix (1× NEB CutSmart, 0.4 U/μL PvuI-HF enzyme), designed to cleave 3′ fragments carrying a PvuI recognition site. The mix was incubated for 1 h at 37 °C, then washed three times in TNT. Finally, to elute DNA, beads were resuspend in 30 µL ddH2O and incubated 10 minutes at 70°C. Beads were then immediately bound to magnet and the supernatant was collected. To remove short fragments,  Ampure beads (Beckman Coulter) were used at 1.8× volume and eluted in 30 µL.
GEO Accession
GSM1477079