GSM2525246: 1772117090_H12; Mus musculus; RNA-Seq
Mice were sacrificed by an overdose of Isoflurane or Ketamin/Xylazine, followed by perfusion through the left ventricle with artificial cerebrospinal fluid, equilibrated in 95%O2/5%CO2 on ice or 4°C. The dentate gyrus was then microdissected from 300 μ m vibratome brain sections. Single-cell suspensions were prepared using the Papain kit (Worthington) with 25-35min enzymatic digestion followed by manual trituration using a BSA-coated p1000 pipette or fire-polished Pasteur pipettes. For hGFAP-GFP mice, GFP-positive cells were sorted on a BD FACSAria II into oxygenated aCSF at 4°C. Cells were loaded at 500-800 cells/μ l at 4° on the C1 Fluidigm medium-sized chip. 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 2x BWT (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween-20) and resuspended in 2 ml 2x 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 (1x 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 1.8x volume and eluted in 30 µL.