GSM3178936: Sample_H3D, hippocampal neuron; Mus musculus; RNA-Seq
A micropipette with a closed, tapered end was used to sever dendrites from the cell body. Another micropipette was used to aspirate the soma, which was deposited into a tube containing first strand synthesis buffer and RNase inhibitor and placed on ice. A separate micropipette was used to aspirate the dendrites, which were deposited into a separate tube as above. Samples were transferred to -80ºC within 30 minutes and stored there until first strand synthesis. Poly-adenylated RNA was amplified using two or three rounds of the aRNA in vitro transcription-based amplification method, as described in (Morris, Singh, and Eberwine 2011). The quality and quantity of the amplified RNA was verified using a Bioanalyzer RNA assay. Strand-specific sequencing libraries were prepared using the Illumina TruSeq Stranded kit according to the manufacturer's instructions, except that the initial poly-A capture step was skipped because the aRNA amplification procedure already selects for poly-adenylated RNA. Samples were sequenced on a HiSeq (100bp paired-end) or NextSeq (75bp paired-end) to an average depth of 25 million reads.