title: GSM6402085: Naïve Old F7; Mus musculus; RNA-Seq
library_construction_protocol: For the contusion injury, mice were anesthetized with ketamine (100 mg/kg)-xylazine (10 mg/kg) mixture. A 15-mm midline skin incision was made, and the connective tissue and muscles were bluntly dissected to expose T6-T13. Laminectomy was performed at T9 to expose the dorsal spinal cord, and the vertebral column was stabilized with angled clamps attached to the T7 and T12 transverse processes. The contusion injury was delivered using an Infinite Horizon Impactor (IH-0400 Impactor) with a force of 90 dynes. After injury, the muscles and skin were sutured separately. Mice were humanely euthanized with an intraperitoneal injection of a lethal dose of Euthasol® (10% v/v), followed by transcardiac perfusion of PBS with 5 U/mL of Heparin. Vertebral columns were obtained. The ventral bone of the vertebrae was cut laterally and was removed to expose the spinal cord. Nervous tissue was carefully dissected from the bone using fine forceps to release the dorsal root ganglia surrounded by the meninges. The meninges was cut along the ventral side and peeled from the spinal cord. Spinal cord meninges was enzymatically digested in RPMI containing 1 mg/mL collagenase VIII, 0.5 mg/mL DNase I, and 2% FBS for 30 minutes at 37°C with shaking. Samples were passed through a 70 µm filter to obtain single-cell suspensions, washed with FACS buffer, and pelleted at 380 xg for 5 minutes. Cells were resuspended in FACS buffer with 1:100 CD16/32 Fc block. They were stained with CD31, CD45, Podoplanin and Lyve1, washed, and resuspended in DAPI-containing FACS buffer. Single meningeal lymphatic endothelial cells were sorted as Live (DAPI-) CD45- CD31+ PDPN+ Lyve1+ cells using a FACS Aria II. Individual cells were sorted into 96-well plates containing 2 µL of 10X RNA lysis buffer (Takara) and 5% RNAse out (Takara) and rapidly frozen over dry ice. Library preparation was performed with 2 µL of single cell lysates arrayed in 96-well PCR plates. ds-cDNA was prepared using a protocol adapted from the Takara-Clontech SMARTer methods. cDNA was blunt ended, had an A base added to the 3' ends, and had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified using a standard Illumina i7 primer to introduce an index sequence and FACSseq Lib PCR 1.0 specific to fragments containing the cell barcode added during cDNA synthesis. Fragments were sequenced on an Illumina NovaSeq.