GSM4368676
GSM4368676: Control Ptf1aCRE/+;Ai14; Mus musculus; RNA-Seq
Nuclei from spinal cords were isolated as described in Matson, K.J.E., Sathyamurthy, A., Johnson, K.R., Kelly, M.C., Kelley, M.W., and Levine, A.J. (2018). Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing. J Vis Exp. PMCID: PMC6235529  DOI:10.3791/58413 10X Genomics Next GEM Single cell 3' Reagent Kit v3.1.  Nuclei were loaded with Single Cell 3' Gel Beads into a Next GEM Chip G and run on the Chromium™ Controller. GEM emulsions are incubated and then broken.  Silane magnetic beads are used to clean up GEM reaction mixture. Read 1 primer sequence is added during incubation and full-length, barcoded cDNA is then amplified by PCR after cleanup. Sample size is checked on the Agilent Tapestation 4200 using the DNAHS 5000 tape and concentration determined by the Qubit 4 Fluorometer (ThermoFisher) using the DNA HS assay. Samples are enzymatically fragmented and undergo size selection before proceeding to library construction.  During library preparation, Read 2 primer sequence, sample index and both Illumina adapter sequences are added.  Subsequently, samples are cleaned up using Ampure XP beads and post library preparation quality control is performed using the DNA 1000 tape on the Agilent Tapestation 4200. Final concentration is ascertained using the Qubit 4 Fluorometer (ThermoFisher) DNA HS assay. Samples are loaded at 1.6 pM and run on the Illumina NextSeq500 High Output Flowcell using V2.5 chemistry.  Run configuration is 28x98x8.
GEO
GSM4368676
GEO Accession
GSM4368676