METHODS FOR DETERMINING SPATIAL AND TEMPORAL GENE EXPRESSION DYNAMICS DURING ADULT NEUROGENESIS IN SINGLE CELLS

摘要

Provided herein are methods of recovering single nuclei from a tissue sample comprising chopping or dounce homogenizing the tissue sample in a nuclear extraction buffer at 4° C. to produce a tissue homogenate; centrifuging the tissue homogenate to produce a nuclear pellet; resuspending the nuclear pellet in a nuclear resuspension buffer comprising bovine serum albumin, RNase inhibitor, and salts to produce a resuspension; and filtering the resuspension through a strainer, wherein the single nuclei are present in a supernatant passed through the strainer. The invention also provides a method of single cell sequencing comprising extracting nuclei from a population of cells under conditions that preserve a portion of the outer nuclear envelope and rough endoplasmic reticulum; sorting single nuclei into separate reaction vessels; extracting RNA from the single nuclei; generating a cDNA library, whereby gene expression data from single cells are obtained. The tissue sample may be fresh or frozen.