GSM5904812: A.25_S2; Mus musculus; RNA-Seq
protocol 1: Mice were euthanized with 1.5x the surgical dose of ketamine and xylazine then perfused transcardially with DEPC buffer until the liver and blood were cleared. A 1 cm piece of spinal cord (centered on the T9 laminectomy site) was quickly dissected and homogenized in 1 ml of TRIzol reagent (ThermoFisher). RNA was extracted according to the manufacturers instructions. Briefly, the tissue lysate was centrifuged at 4°C for 5 mins at 12 000 g. The supernatant was incubated for 5 mins to dissociate nucleoproteins. Samples were incubated with 0.2 ml of chloroform for 2.5 mins then shaken vigorously for 15 sec.  Samples were centrifuged at 4°C for 15 mins at 12 000 g. The aqueous phase was collected and mixed with 0.5 ml of isopropanol with 2 ul of glycogen then stored at -20°C. Samples were centrifuged (4°C, 5 mins, 12 000 g) and the pellet resuspended in 75% ethanol. The sample was vortexed, centrifuged (4°C, 5 mins, 7 5 00 g), the supernatant removed and pellet dried for 5-10 mins. The pellet was resuspended in 20 μl RNAse free water and warmed to 55°C. RNA yield was determined using Nanodrop. RNA was purified using the QIAGEN RNeasy spin column steps according to manufacturers instructions. RNA quality and final yield was then measured using a RNA 6000 NanoAssay by Chip. Samples with RIN > 7 were included for analysis (average RIN = 8.6). Samples were stored at -80°C until sequencing. protocol 2: Mice were euthanized with 1.5x the surgical dose of ketamine and xylazine. A 1 cm piece of unperfused, fresh spinal cords (centered on the T9 laminectomy site) was quickly dissected. Tissue was dissociated to a single cell suspension using a gentleMACS dissociator (Miltenyi) and mouse adult brain dissociation kit (Miltenyi, 130-107-677). Dissociated cells were passed through a sterile 70 μm filter to remove large particulate debris. Myelin debris was removed using myelin removal beads (Miltenyi, 130-096-733). Dead cells were removed using Viahance dead cell removal (BioPAL, #CP-50VQ02). Cells were diluted to 1000 cells/μl. protocol 1: RNA-seq was carried out by the UCLA Technology Center for Genomics and Bioinformatics. The library was prepared using Nugen Ovation RNA Ultra Low Input (500 pg) + Kapa Hyper. The quantity and quality of cDNA were assessed using an Agilent 2100 Expert High Sensitivity DNA Assay. cDNA samples were sequenced at UCLA Technology Center for Genomics and Bioinformatics. Reads were aligned to the mouse GRCm38 reference genome using STAR (v.2.4.0). Read counts for RefSeq genes (mm10) were generated by HTSeq v.0.6.1. Low-count genes were filtered and fragments per kilobase per million mapped reads (FPKM) values were generated. protocol 2: The single cell suspension and Chromium Single Cell 3' V3 reagents (10X Genomics) were loaded into chips to collect individual cells encapsulated by gel beads in emulsion (GEMs) using the 10X Chromium Controller. cDNA and library amplification for an optimal signal was 13-16 cycles. Sequencing was conducted on HiSeq4000 (Novogene).