GSM7719122: anti-gD 200mpk day3 rep 3; Macaca fascicularis; RNA-Seq
Frontal cortex samples from cynomolgus monkey were placed in the 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 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 tissues 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 tissues were not allowed to thaw before placing in the lysis buffer and douncing. From this homegenate, nuclei were isolated density-gradient centrifugation. The homogenate was layered over an Optiprep density (40%, 20% and 10% for primates) gradient and centrifuged at 2500 g for 20 min. Nuclei at the 20/40 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. 10x Genomics 3' v3 chemistry