GSM7850408: HPeV3_SIDS_run_1_neun_pos; Homo sapiens; RNA-Seq
Approximately 150-300μm of fresh frozen (non-fixed) brainstem tissue was cryosectioned at the level of the obex + 2mm (as identified by human brainstem atlas landmarks, consistent with our previous studies), a region containing both the raphe obscurus and raphe palladus, where 5-HT neuron cell bodies reside, as well as other nuclei implicated in cardiorespiratory regulation. Tissue sections were transferred to a 1.5ml Eppendorf tube on dry ice. Tissue was then homogenized and nuclei extracted using the Nuclei Isolation Kit:Nuclei EZ prep. Harvested nuclei were then incubated in NucBlue live cell stain for 20 minutes at room temperature, sorted on the On-Chip microfluidic sorter to eliminate tissue debris and other contaminants, and then quantified using a hemacytometer. Note: for two sample runs – SIDS2_run_1 and HPeV3_SIDS_run_1 – nuclei were additionally stained with NeuN, and NeuN positive and negative nuclei were separately sorted and sequenced (in an attempt to enrich for neuronal nuclei in the NeuN positive condition; library name suffixes indicate NeuN positive or negative condition). For all other sample runs, only NucBlue staining was performed. Harvested nuclei were gently centrifuged and suspended to an appropriate concentration (~700-1200 nuclei/μl) and processed through the 10X Genomics Chromium Single Cell 3' v3 protocol followed by Illumina NextSeq 500 sequencing.