title: GSM5343188: PIlot_sample 9_preBötC inspiratory neuron; Mus musculus; RNA-Seq
library_construction_protocol: Once the electrical phenotype of preBötC inspiratory neurons was determined, the cellular contents of the neuron were extracted by applying gentle negative pressure (0.7-1.5 psi). The patch pipette was quickly retracted, and the extracted cellular contents were ejected by breaking the tip of the pipette at the bottom of the RNase-free PCR tube containing 4 µL nuclease-free water with 2% RRI. The sample was briefly spun, snap-frozen in liquid nitrogen and stored at -80˚C. The extracted RNA from patch-clamped neurons was converted to cDNA according to SMART-Seq v4 ultra low input RNA kit for Sequencing protocol.  First strand cDNA was synthesized by performing reverse transcription in a thermal cycler (42 °C for 90 min, 70 °C for 10 min). The cDNA was then amplified by at 95 °C for 1 min, 17 cycles of 98 °C for 10 s, 65 °C for 30 s, 68 °C for 3 min; 72 °C for 10 min. PCR-amplified cDNA was purified by immobilization on AMPure beads, which were then washed with 80% ethanol and eluted. Amplified cDNA was validated using the Agilent 2100 Bioanalyzer and Agilent's High Sensitivity Kit (5067-4626, Agilent Technologies, CA), and its concentration was determined using Qubit dsDNA High-sensitivity Assay Kit (Molecular Probes). The full-length cDNA output was processed with the Nextera Library Preparation Kits (FC-131-1024, Illumina, San Diego, CA) to obtain cDNA libraries for RNA-Seq experiments. The cDNA libraries were sequenced using an Illumina HiSeq 4000 Sequencing system with paired end (150 bp) reads. Investigators were blinded to cell type during library construction and sequencing.
attributes.tag: GEO Accession
attributes.value: GSM5343188