Transgenic mice were used to fluorescently label various populations of hippocampal principal cells. Animals were sacrificed between P25-P32, and manual sorting was performed to purify for fluorescent neurons from microdissected brain slices. Approximately 100 cells were obtained per animal, and three animals (i.e., three biological replicates) were obtained for each dataset.
For library preparation, total RNA was isolated, ERCC spike-in controls were added, and cDNA was amplified from this input material. Sequencing libraries were constructed from the amplified cDNA, and four barcoded libraries were pooled per sequencing lane on a HiSeq 2500. The single-end 100 bp reads generated from sequencing were aligned, quantified, and analyzed with the TuxedoSuite pipeline, which outputs gene expression as FPKM (Fragments Per Kilobase of Exon Per Million Reads Mapped) and assesses differential expression with a false discovery rate cutoff (typically 5%).
For additional details, see:
Cembrowski, M.S., Wang., L., Sugino, K., Shields, B.C., Spruston, N.Hipposeq: a comprehensive RNA-seq database of gene expression in hippocampal principal neurons.
eLife 5, 10.7554/eLife.14997 (2016).