GSM6069078: mouseDRG_sample10; Mus musculus; RNA-Seq
GSM6069078
DRGs from lumbar levels from both right and left sides were collected in the ice-cold lysis buffer (20 mM NaCl, 5 mM MgCL2, 0.1% TX-100, 10 mM Tris-HCl pH7.2) containing EDTA-Free protease inhibitor, RNAse inhibitor, and RNase-free DNase. Dounce homogenization was used to dissociate all DRG tissues. Before douncing, 1 mL HBSS containing 3% BSA Fraction VI and RNAse inhibitor in nuclei suspension buffer (NSB) was added to the  2 mL Dounce Tissue Grinder. The DRGs were homogenized with an A (“loose”) pestle using 5 to 10 strokes. The homogenate was then filtered through a 70 micron filter and spun down. The pellet was resuspended in the NSB. The frozen DRGs were not allowed to thaw before placing in the lysis buffer and douncing. From this homegenate, nuclei were isolated using two different methods: density-gradient centrifugation and FACS. For density-gradient centrifugation method, the homogenate was layered over an Optiprep density (35%, 16%, 8% for rodents) gradient and centrifuged at 2500 g for 20 min. Nuclei at the 16/35 interface were aspirated and mixed with an equal volume of NSB. Nuclei were re-pelleted, washed, and counted using a hemocytometer prior to loading into a 10X Genomics Chromium controller. For FACS, nuclei from the density gradient were pelleted by centrifugation, labeled with propidium iodide and DAPI, and sorted using the Aria Fusion Flow Cytometer. Nuclei were selected based on double labeling with DAPI and PI and sorted into eppendorf tubes containing 0.5 mL nuclei suspension buffer (NSB).  Nuclei were then counted, pelleted and resuspended prior to loading into a 10X Genomics Chromium controller. Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (10X Genomics) were used for library preparation according to the manufacturer's user guides. The Cell-RT mix was prepared to aim for 10,000 nuclei per sample and applied to the ChromiumTM Controller for GEM generation and barcoding. Then samples were subjected to post GEM-RT cleanup, cDNA amplification (11 cycles with v3.1), and library construction according to the user manual. Sample index PCR was done with 12 cycles. Libraries were then quantified by Qubit dsDNA HS Assay Kit and profiled by Bioanalyzer High Sensitivity DNA kit.