title: GSM8878095: Hind limb non-CSNs, layer 5 M1, replicate 1; Mus musculus; RNA-Seq
library_construction_protocol: Adult intact cell isolation was done following our previously published protocol (Golan and Cafferty, 2021). Briefly, mice that received retrograde injections into either the lumbar or cervical cord were anesthetized with isoflurane and transcardially perfused with artificial cerebrospinal fluid (aCSF) ten days after retro-AAV injection.  The brain was dissected and submerged in ice-cold carbogenated aCSF for three minutes before being transferred to a brain matrix (Braintree Scientific, Inc.) where four 500 µm sections through M1 were sliced and sections were transferred to a petri dish containing ice cold carbogenated aCSF. Regions of M1 (+1.0 to -0.5 mm relative to Bregma) containing labeled CSNs were dissected under a fluorescent microscope and transferred to a 5 ml Eppendorf tube with dissociation buffer with papain. The sample in dissociation buffer with papain was incubated at 34℃ for 70 minutes on a shaker at medium speed. After 70 minutes, the dissociation solution with papain was replaced with a Stop buffer. The Stop solution was then replaced with 800 µl of dissociation buffer and triturated with Pasteur pipettes with decreasing diameters (600 µm, 300 µm and 150 µm) and placed on ice. To differentiate between intact neurons and nuclei, a cell-permeable dye was added at a concentration of 1:1000 for 5 minutes (Invitrogen, L34974). The sample was then centrifuged at 300 g for 10 minutes at 4℃ and resuspended in 500 µl of dissociation buffer. Fluorescent single cells were isolated from rbp4 cre: Ai14 mice and C57Bl/6J mice injected with retro-AAV-CAG-GFP.  Sorting was performed on the Sony SH800 sorter with a 130 µm nozzle, ensuring a sheath pressure and sample pressure of less than 9 PSI. To ensure pyramidal neurons were sorted, size reference beads were used to set a minimum diameter of 10 µm (Spherotech, PPS-5 and PPS-6) for all collected cells. To exclude non-intact cells, the cells were sorted for the presence of the cell-permeable dye. A sample of non-fluorescent tissue from adjacent regions of the cortex was used as negative control to set the gate for the presence of fluorescence (GFP or tdTomato). The sample was then sorted into Eppendorf tubes containing 4℃ dissociation buffer and immediately sequenced at the Yale Center for Genome Analysis (YCGA). Library construction was perfomed according to manufacturers instructions. Single cell suspension in RT Master Mix was loaded on the Single Cell A Chip and partitioned with a pool of about 750,000 barcoded gel beads to form nanoliter-scale Gel Beads-In-Emulsions (GEMs). Each gel bead has primers containing (i) an Illumina® R1 sequence (read 1 sequencing primer), (ii) a 16 nt 10x Barcode, (iii) a 12 nt Unique Molecular Identifier (UMI), and (iv) a poly-dT primer sequence (30nt). Upon dissolution of the Gel Beads in a GEM, the primers were released and mixed with cell lysate and Master Mix. Incubation of the GEMs then produced barcoded, full-length cDNA from poly-adenylated mRNA.Silane magnetic beads were used to remove leftover biochemical reagents and primers from the post GEM reaction mixture. Full-length, barcoded cDNA was then amplified by PCR to generate sufficient mass for library construction. Enzymatic Fragmentation and Size Selection were used to optimize the cDNA amplicon size prior to library construction. R1 (read 1 primer sequence) was added to the molecules during GEM incubation. P5, P7, a sample index, and R2 (read 2 primer sequence) were added during library construction via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers were used in Illumina bridge amplification. The Single Cell 3' Protocol produces Illumina-ready sequencing libraries. A Single Cell 3' Library comprises standard Illumina paired-end constructs which begin and end with P5 and P7. The Single Cell 3' 16 bp 10x Barcode and 12 bp UMI are encoded in Read 1, while Read 2 is used to sequence the cDNA fragment (91bp). Minimum sequencing depth is 20,000 read pairs per cell.
identifiers.namespace: GEO
identifiers.id: GSM8878095_r1