title: GSM1927657: 1772089194_H02; Mus musculus; RNA-Seq
library_construction_protocol: Mice were deeply anesthetized (5% isoflurane) and transcardially perfused with 40 ml ice-cold pre-oxygenated (95% O2/5% CO2) cutting solution containing (in mM): 90 NaCl, 26 NaHCO3, 2.5 KCl, 1.2 NaH2PO4, 10 HEPES-NaOH, 5 Na-ascorbate, 5 Na-pyruvate, 0.5 CaCl2, 8 MgSO4 and 20 glucose. A central column of the mouse hypothalamus spanning the preoptic area-Arc (rostrocaudal axis), paraventricular nucleus (dorsal) and the ventrolateral hypothalamic area (lateral) was microdissected from serial 300-micrometer-thick coronal slices under microscopy guidance and dissociated using the Papain Dissociation System (Worthington). Isolated single cells were concentrated by centrifugation to a density of 600-1,000 cells/?microlitre. After mixing C1 suspension reagent (4 microlitre; "C1 reagents", Fluidigm Inc.) to the cell suspension (7 microlitre), this mixture was loaded into a C1-AutoPrep IFC microfluidic chip (Fluidigm Inc.) 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.
attributes.tag: GEO Accession
attributes.value: GSM1927657