GSM6529550: Supralumbar_Uninjured_biol_rep1; Mus musculus; RNA-Seq
Mice were anesthetized with isoflurane and promptly decapitated. The brain and brainstem were dissected out and placed in ice-cold slushy artificial cerebrospinal fluid (Hearing et al., 2013) for one minute. Brains were then sectioned in the sagittal plane at 500-micron intervals using Adult Mouse Brain Slicer Matrix on ice (Zivic Instruments BSMAS005-2). A total of nine sections were created from each animal including a midline section and four sections moving lateral into each hemisphere. Neurons retrogradely labelled from lumbar spinal cord were then micro dissected using a stereomicroscope and fluorescence adapter (NIGHTSEA SFA-GR).  The three sections from the left hemisphere and one midline section were dissected and flash frozen in a 1.5mL Eppendorf DNA LoBind Microcentrifuge tube. The three sections from the right hemisphere were then collected and flash frozen with identical conditions, as mentioned above. Samples were then stored at -80 °C until FACS sorting. To prepare cell nuclei on the day of library preparation frozen tissue was promptly transferred from -80 °C storage to a chilled 2mL dounce with chilled Nuclei EZ Lysis Buffer (Sigma-Aldrich N 3408). Sample was dounced 25 times with pestle A and 20 times with pestle B. The dounced sample was then transferred to a chilled 15mL conical tube and an additional 2mL of Nuclei EZ Lysis Buffer was added and gently mixed by inversion. The sample incubated on ice for 5 minutes and then was centrifuged at 500G at 4 °C. The supernatant was removed, and the pellet was resuspended in 4mL of Nuclei EZ Lysis Buffer. The sample again was incubated on ice and centrifuged at 500G at 4 °C for 5 minutes. Following the centrifugation, the supernatant was removed, and the pellet resuspended in 500uL of Nuclei Suspension Buffer (2% BSA, 40U/uL RNase Inhibitor (Invitrogen Ref. AM2684), 1X PBS). The resuspended solution was then filtered through a 20um filter and moved directly to the FACS machine. The collection tube for the sorted nuclei was coated with 5% BSA and contained the 10X Genomics master mix. We initially gate out debris using side scatter area (SSC-A) vs forward scatter area (FSC-A) and forward scatter width (FSC-W) vs forward scatter area (FSC-A). To eliminate doublets, the nuclei that pass through the gates above were then gated by side scatter width (SSC-W) vs side scatter height (SSC-H). Nuclei that passed through all the above gates were then filtered by level of fluorescence so that only the brightest were collected. With the above parameters, the FACS machine was set to collect 5,000 nuclei in five to seven minutes. Sample libraries were prepared according to Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (PN-1000269), according to the manufacture protocol (10X Genomics, CG000204 Rev D).