BioProject

To isolate Cx3cr1 fate mapped cells following lysolecithin-induced demyelination in the spinal cord, we performed stereotaxic injection of lysolecithin into the ventral white matter tract of the thoracic spinal cord in adult Cx3cr1-CreER:Rosa26-tdTomato+ve mice treated with tamoxifen 4 weeks previously. More...

To isolate Cx3cr1 fate mapped cells following lysolecithin-induced demyelination in the spinal cord, we performed stereotaxic injection of lysolecithin into the ventral white matter tract of the thoracic spinal cord in adult Cx3cr1-CreER:Rosa26-tdTomato+ve mice treated with tamoxifen 4 weeks previously. We FACS collected viable tdTomato+ve cells from uninjured mice and from mice at 5 days post-injury. All samples were processed according to 10X Genomics ChromiumTM Single Cell 3’ Reagent Guidelines v2 Chemistry as per the manufacturer’s protocol. In brief, single cells were sorted into 1% BSA–PBS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. Sequencing was performed first using Illumina MiSeq SR50 to approximate the number of recovered cells in each sample. We recovered 705 and 580 cells for Uninjured and Injured conditions, respectively. Based on this, we determined lane distributions for sequencing using Illumina HiSeq 4000 PE (75 bp paired-end reads) with a targeted sequencing depth of ~100,000-150,000 reads/cell. Overall design: Single-cell RNA-seq of Uninjured and injuired spinal cord Cx3cr1 fate mapped cells Less...