GSM7893568: SM-J39SU_S050_E1-50; Mus musculus; RNA-Seq
Single nuclei were isolated by adapting a protocol from the Allen Institue for Brain Science available at protocols.io (dx.doi.org/10.17504/protocols.io.bq7emzje). Spinal projecting neurons (SPNs) were enriched from the nucleus suspensions using fluorescence activated nucleus sorting (FANS). For 10x, single SPN nuclei were sorted using a BD FACSARIA II with a 70 µm custom pressure nozzle (50 psi). Briefly, single nuclei were captured by sorting on 'four-way-purity mode' and gating on DAPI-positive while excluding debris and aggregates, then gating on GFP and/or mScarlet signal. Nuclei were sorted (to a maximum number of 16,000 nuclei) using two-way sorting (GFP-positive separate from mScarlet- and double-positive) into PCR tubes (pre-coated with 5% BSA overnight) containing 20uL of sort buffer. After sorting, PCR tubes were briefly centrifuged and then placed on ice until proceeding with the 10x platform. For SSv4, single nuclei were sorted using a Sony SH800 Cell Sorter or MA900 Multi-Application Cell Sorter using a 100 µm chip. Briefly, single nuclei were sorted on 'single-cell' mode and captured by gating on singlet DAPI-positive, then gating on GFP and/or mScarlet signal to specifically sort cervical and/or lumbar project SPNs, respectively. Indexed plate-based sorting was used to sort GFP-, mScarlet, and double-positive nuclei into strip tubes containing 11.5uL of SMART-Seq v4 lysis buffer. Lysed FANS-sorted nuclei were then briefly centrifuged, frozen on dry ice, and stored at -80ºC until proceeding with the SSv4 platform. 10x processing was performed using the Chromium Next GEM Single Cell 3ʹ Kit v3.1 (1000268, 10x Genomics). To optimize yield, sorted nucleus suspensions were loaded directly into the 10x Chromium Controller without post-FANS spin down and re-suspension. Reverse transcription, cDNA amplification, and library construction were performed according to the manufacturer's protocol. Libraries were sequenced on an Illumina NovaSeq 6000, targeted at a sequencing depth of 120,000 reads per nucleus. SMART-Seq v4 (SSv4) processing was performed according to previously established procedures47 available at protocols.io (dx.doi.org/10.17504/protocols.io.8epv517xdl1b/v2). Briefly, the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (634894, Takara) was used to reverse transcribe poly(A) RNA and amplify full-length cDNA. Samples were amplified for 16-21 cycles in eight-well strips. Library preparation was then performed using Nextera XT DNA Library Preparation (FC-131-1096, Illumina) with a custom index set (Integrated DNA Technologies) according to the manufacturer's instructions with modifications to reduce the volumes of all reagents and cDNA input to 0.2x of the original protocol. Libraries were sequenced on either an Illumina NovaSeqSP-XP or an Illumina NextSeq2000, targeting 500,000 reads per nucleus. Single-nucleus RNA sequencing (snRNAseq)