3-day post injury, rep4
To capture, label, and generate transcriptome libraries of individual cells we used the 10X genomics Chromium Single Cell 3 Library and Gel Bead Kit v2 (Cat #PN-120237) following the manufactures protocols. Briefly, the single cell suspension, RT PCR master mix, gel beads, and partitioning oil were loaded into a Single Cell A Chip 10 genomics chip, placed into the chromium controller, and the chromium Single Cell A program was run to generate GEMs (Gel Bead-In-EMulsion) that contain RT-PCR enzymes, cell lysates and primers for illumine sequencing, barcoding, and poly-DT sequences. GEMs are then transferred to PCR tubes and the RT-PCR reaction is run to generate barcoded single cell identified cDNA. Barcoded cDNA is used to make sequencing libraries for analysis with Illumina sequencing. We captured 1709 cells from young uninjured, 3459 from the aged uninjured, 5077 from the 4-d post injury and 2668 from the 7-d post injury. Sequencing was completed on an Illumina NovaSeq 6000, using paired end 150 cycle 2x150 reads by the genomics and microarray core at the University of Colorado Anschutz Medical Campus.