US 12,221,720 B2
Methods for determining spatial and temporal gene expression dynamics during adult neurogenesis in single cells
Naomi Habib, Cambridge, MA (US); Aviv Regev, Cambridge, MA (US); Eugene Drokhlyansky, Cambridge, MA (US); Anindita Basu, Cambridge, MA (US); Inbal Avraham-Davidi, Cambridge, MA (US); Orit Rozenblatt-Rosen, Cambridge, MA (US); and David A. Weitz, Cambridge, MA (US)
Assigned to The Broad Institute, Inc., Cambridge, MA (US); Massachusetts Institute of Technology, Cambridge, MA (US); and President and Fellows of Harvard College, Cambridge, MA (US)
Appl. No. 16/763,681
Filed by The Broad Institute, Inc., Cambridge, MA (US); Massachusetts Institute of Technology, Cambridge, MA (US); and President and Fellows of Harvard College, Cambridge, MA (US)
PCT Filed Nov. 13, 2018, PCT No. PCT/US2018/060860
§ 371(c)(1), (2) Date May 13, 2020,
PCT Pub. No. WO2019/094984, PCT Pub. Date May 16, 2019.
Claims priority of provisional application 62/734,988, filed on Sep. 21, 2018.
Claims priority of provisional application 62/723,425, filed on Aug. 27, 2018.
Claims priority of provisional application 62/585,529, filed on Nov. 13, 2017.
Prior Publication US 2021/0395821 A1, Dec. 23, 2021
Int. Cl. C40B 50/06 (2006.01); A61K 35/30 (2015.01); C12N 5/0793 (2010.01); C12N 5/0797 (2010.01); C12Q 1/6806 (2018.01); C12Q 1/6881 (2018.01); C40B 30/06 (2006.01); C12N 5/00 (2006.01)
CPC C40B 30/06 (2013.01) [A61K 35/30 (2013.01); C12N 5/0619 (2013.01); C12N 5/0623 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6881 (2013.01); C12N 5/0081 (2013.01); C12Q 2600/158 (2013.01)] 13 Claims
OG exemplary drawing
 
1. A method of producing a temporally phased single-cell sequencing library comprising cells along a continuous trajectory of adult neurogenesis in the adult brain, the adult dorsal ganglion (DG) or in the adult spinal cord, said method comprising:
(a) treating more than one population of neurogenic cells of heterogeneous cell types or subtypes, with a nucleoside analogue, wherein the neurogenic cell is selected from the group consisting of: a neuronal stem cell, a neuronal precursor cell, a neuroblast, an immature neuron and a newborn neuron, and wherein the nucleoside analogue is incorporated into replicating DNA and is configured for labeling with a detectable marker;
(b) isolating a first population of neurogenic cells at one time point and isolating at least one other population of neurogenic cells at a later time point;
(c) staining the nucleoside analogue incorporated into replicating DNA with the detectable marker within each population of neurogenic cells, wherein the replicating DNA is stained with the detectable marker;
(d) sorting the stained and/or unstained neurogenic cells; and
(e) sequencing the RNA from the sorted single neurogenic cells, whereby single cell gene expression data is obtained for neurogenic cells at different stages of neurogenesis.