GSM8551856: Naïve, rep 1; Mus musculus; RNA-Seq
To visualize the lesion, mice were injected with 1% neutral red solution (500µL, intraperitoneal, prepared in PBS), approximately 2 hours prior to intracardiac perfusion with PBS. The spinal cord was extracted from perfused mice. Lesions demarcated with neutral red, rostral nonlesioned white matter tissue, and naive spinal cord white matter tissue were microdissected and collected according to experimental batch. Each lesioned and nonlesioned sequencing sample is the result of 3-4 microdissected  pooled  samples from lesioned mice ensuring large biological variability.  Single nucleus suspensions were created utilzing the Nuclei PURE lysis kit (Signma #646563),  with a sucrose density gradient and ultracentrifugation (30,000 rcf, 15,600 rpm). Nuclei pellets were washed, filtered at 40µm, and visualized under brightfield for intact nucleus morphology. Sequencing libraries were prepared according to library batch number, following the protocol for 10x Genomics Chromium next GEM Single Cell 3' v3.1 Dual Index. Briefly, 10,000 nuclei per sample was loaded into Chromium Single Cell 3' Chip G wells and placed in the Chromium controller for GEM generation as outlined in 10x Genomics User Guide (CG000315. Rev A) for a targeted recovery of 5,000 nuclei per sample. GEMs were incubated in the thermal cycler for cDNA barcoding, followed by GEM-RT cleanup and cDNA amplification using v3.1 Chromium GEM kit. Total cDNA concentration was measured by Qubit dsDNA HS and 25% of the total cDNA was used for 3' gene expression library construction using Dual Index Kit TT Set A. The resulting sequencing libraries was assessed for quality using the BioAnalyzer 2100 with a High Sensitivity DNA kit (Agilent), and quantified using the KAPA Library Quantification Kit, Illumina Platforms (KAPA Biosystems) according to manufacturer's protocol. Libraries were sequenced on a S4 flow cell to an average depth of 400 million reads per sample.