title: GSM7876110: Telencephalon+POA, male, snRNA-seq, rep1; Amphiprion ocellaris; RNA-Seq
library_name: GSM7876110
library_construction_protocol: Frozen telencephalon tissue samples were incubated on ice for 30 minutes in chilled lysis buffer (10 mM Tris-HCL, 10 mM NaCl, 3 mM MgCl2, 1% Nonidet P40 Substitute, and Nuclease-free H2O) with gentle rotation. Following lysis, HAB medium (with 2% B27 and 0.5 mM Glutamax) was added and tissue was triturated using silanized glass Pasteur pipettes (500 μm internal diameter) to complete tissue dissociation. Samples were centrifuged (600rcf, 5 minutes, 4°C) and resuspended in chilled wash and resuspension buffer (1X PBS, 2% BSA, 0.2 U/μl RNase Inhibitor). Nuclei suspensions were filtered through 40 μm Flowmi® cell strainers and 30 μm MACS® SmartStrainers to remove debris and large nuclei aggregations. Finally, fluorescence activated cell sorting (FACS) was used to further purify samples. DAPI (1.0 μg/ml) was used as a nuclear stain and viable, intact nuclei singlets were selected and sorted into collection buffer (1X PBS, 2% BSA, 0.2 U/μl RNase Inhibitor) using a BD FACSAriaTM Fusion Cell Sorter. Libraries were prepared according to manufacturer instructions (Chromium Single Cell 3' Reagent Kits User Guide v3.1 Chemistry (10X Genomics)). Single nuclei suspensions were loaded onto the 10x Genomics Chromium Controller and partitioned into individual GEMs (gel bead-in emulsion), each containing a single nucleus, a barcoded gel bead and master mix with reverse transcription (RT) reagents. Primers released from each gel bead include a 10X barcode, unique molecular identifier (UMI) and Illumina R1 primer. Partitioned nuclei were lysed and GEMs were incubated to produce full-length, barcoded cDNA. Barcoded cDNA were purified using Silane magnetic beads and amplified via PCR . Amplified cDNA was size-selected with SPRIselect. During final library construction, an Illumina R2 primer sequence, I5 and I7 paired-end construct sequences, and a sample index were added. Quality was assessed using high sensitivity DNA analysis on the Bioanalyzer 2100 system (Agilent) and libraries were quantified using Qubit 2.0 (Invitrogen).