GSM5343173: Sample 12_Dbx1-derived preBötC neuron; Mus musculus; RNA-Seq
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 extracted RNA from patch-clamped neurons was converted to cDNA according to the SMART-seq HT protocol (Takara Bio, USA).  First, samples were denatured at 72 ˚C for 3 min. poly(A)+ RNA was reverse transcribed using a tailed oligo(dT) primer. First strand cDNA synthesis and double-stranded cDNA amplification were performed in a heated thermal cycler using the following program: 42 °C for 90 min; 95 °C for 1 min; 18 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 then purified by immobilization on Agencourt AMPure XP beads (Beckman Coulter, CA), which was then washed with 80% ethanol and eluted with elution buffer. Once full-length cDNA was synthesized, libraries for sequencing were prepared using the SMART-seq PLUS kit (Takara Bio USA, CA), which uses Takara Bio's patented library preparation utilizing stem-lop adapters to construct high-quality libraries. Unique dual indexes were used on the amplified libraries. Quality control was performed on both, the amplified cDNA and the final library using a Bioanalyzer High Sensitivity kit (Agilent, Santa Clara, CA) and Qubit dsDNA High-sensitivity Assay Kit (Molecular Probes, Eugene, OR). cDNA samples containing less than 150 pg/µl cDNA were not sequenced. The cDNA libraries were sequenced using an Illumina HiSeq X Sequencing system with paired end (150 bp) reads. Investigators were blinded to cell type during library construction and sequencing.
GEO
GSM5343173