GSM8864575: SplitSeq_Cortex_GEX; Rattus norvegicus; RNA-Seq
Nuclei from visual cortex were obtained from one rat, anesthetized with isoflurane and sacrificed. The dissected visual cortex tissue was immediately placed in a dry-ice-cold tube for immediate freezing until processing for nuclei isolation. Dissected frozen cortex was resuspended and mechanically homogenized using dounce homogeneizer to release nuclei following the Salty EZ 10 protocol (dx.doi.org/10.17504/protocols.io.bx64prgw). Nuclei were resuspended in 500μL of resuspension buffer and were then fixed with the Evercode Nuclei Fixation kit (#ECF2003) according to manufacturer instructions (Parse Biosciences) and FACS-sorted based on DAPI intensity to remove debris (BD ARIA). Sorted nuclei were finally frozen down. All steps were carried on ice or at 4°C. Nuclei were thawed and loaded in round 1, 2 and 3 plates for combinatorial barcoding according to manufacturer instructions (#UMWT3300 , Parse Biosciences). Six sub-libraries of 12,500 nuclei were prepared to target 75,000 nuclei and 12,000 nuclei per condition. Libraries were sequenced using the Novaseq 6000 platform (Illumina). Libraries were prepared according to Parse Biosciences version 3 chemistry & Illumina protocols