GSM2160424: C1-1771051-150-C04; Mus musculus; RNA-Seq
Selected CNS regions were dissected in 300µm thick vibratome sections and cut out with scalpel. Tissue was then dissociated into a single cell suspension (described in Zeisel & Munoz-Manchado, Science 2015 Mar 6;347(6226):1138). For the spinal cord, an extra gradient step was used after the strainer and final centrifugation step. The pellet was ressuspended in 2ml Neurobasal-A medium including supplements and transferred onto an Optiprep Density Gradient, which consisted of 3 phases (Bottom phase: 160µl Optiprep + 340µl Neurobasal-A; middle phase: 220µl Optiprep + 1780µl Neurobasal-A; top phase: 75µl Optiprep + 925µl Neurobasal-A). The gradient was then centrifuged at 300g for 10min at 8°C and cells were located between the bottom and the middle phase. Cell suspension in a concentration of 600-1000 cells/μL was used. C1 Suspension Reagent was added in a ratio of 4 μL to every 7 μL cell suspension. 11 μL of the cell suspension mix was 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.