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1

Postmortem Brain Tissue Sectioning

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Whole postmortem brain specimens were transported to the Allen Institute on ice. Standard processing of whole brain specimens involved bisecting the brain through the midline and embedding of individual hemispheres in Cavex Impressional Alginate for slabbing. Coronal brain slabs were cut at 1cm intervals through each hemisphere and individual slabs were frozen in a slurry of dry ice and isopentane. Slabs were then vacuum sealed and stored at −80°C until the time of further use.
Middle temporal gyrus (MTG) was identified on and removed from frozen slabs of interest, and subdivided into smaller blocks for further sectioning. Individual tissue blocks were processed by thawing in PBS supplemented with 10mM DL-Dithiothreitol (DTT, Sigma Aldrich), mounting on a vibratome (Leica), and sectioning at 500μm in the coronal plane. Sections were placed in fluorescent Nissl staining solution (Neurotrace 500/525, ThermoFisher Scientific) prepared in PBS with 10mM DTT and 0.5% RNasin Plus RNase inhibitor (Promega) and stained for 5 min on ice. After staining, sections were visualized on a fluorescence dissecting microscope (Leica) and cortical layers were individually microdissected using a needle blade micro-knife (Fine Science Tools).

Hodge R.D., Bakken T.E., Miller J.A., Smith K.A., Barkan E.R., Graybuck L.T., Close J.L., Long B., Johansen N., Penn O., Yao Z., Eggermont J., Hollt T., Levi B.P., Shehata S.I., Aevermann B., Beller A., Bertagnolli D., Brouner K., Casper T., Cobbs C., Dalley R., Dee N., Ding S.L., Ellenbogen R.G., Fong O., Garren E., Goldy J., Gwinn R.P., Hirschstein D., Keene C.D., Keshk M., Ko A.L., Lathia K., Mahfouz A., Maltzer Z., McGraw M., Nguyen T.N., Nyhus J., Ojemann J.G., Oldre A., Parry S., Reynolds S., Rimorin C., Shapovalova N.V., Somasundaram S., Szafer A., Thomsen E.R., Tieu M., Quon G., Scheuermann R.H., Yuste R., Sunkin S.M., Lelieveldt B., Feng D., Ng L., Bernard A., Hawrylycz M., Phillips J.W., Tasic B., Zeng H., Jones A.R., Koch C, & Lein E.S. (2019). Conserved cell types with divergent features in human versus mouse cortex. Nature, 573(7772), 61-68.

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Transcardial Perfusion and Nuclei Isolation for Marmoset M1 Cortex

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Marmoset experiments were approved by, and in accordance with, the Massachusetts Institute of Technology IACUC, protocol number 051705020. Two adult marmosets (2.3 and 3.1 years old; one male, one female; Extended Data Table 2) were deeply sedated by intramuscular injection of ketamine (20–40 mg kg⁻¹) or alfaxalone (5–10 mg kg⁻¹), followed by intravenous injection of sodium pentobarbital (10–30 mg kg⁻¹). When the pedal withdrawal reflex was eliminated and/or the respiratory rate was diminished, animals were transcardially perfused with ice-cold sucrose–HEPES buffer. Whole brains were rapidly extracted into fresh buffer on ice. Sixteen 2-mm coronal blocking cuts were rapidly made using a custom-designed marmoset brain matrix. Coronal slabs were snap-frozen in liquid nitrogen and stored at −80 °C until use.
As for human samples, marmoset M1 was isolated from thawed slabs using fluorescent Nissl staining (Neurotrace 500/525, ThermoFisher Scientific). Stained sections were screened for histological hallmarks of primary motor cortex. Nuclei were isolated from the dissected regions as described (https://www.protocols.io/view/extraction-of-nuclei-from-brain-tissue-2srged6), and were processed using the 10× Chromium single-cell 3′ reagent kit v3. 10× chip loading and sample processing was done according to the manufacturer’s protocol.

Bakken T.E., Jorstad N.L., Hu Q., Lake B.B., Tian W., Kalmbach B.E., Crow M., Hodge R.D., Krienen F.M., Sorensen S.A., Eggermont J., Yao Z., Aevermann B.D., Aldridge A.I., Bartlett A., Bertagnolli D., Casper T., Castanon R.G., Crichton K., Daigle T.L., Dalley R., Dee N., Dembrow N., Diep D., Ding S.L., Dong W., Fang R., Fischer S., Goldman M., Goldy J., Graybuck L.T., Herb B.R., Hou X., Kancherla J., Kroll M., Lathia K., van Lew B., Li Y.E., Liu C.S., Liu H., Lucero J.D., Mahurkar A., McMillen D., Miller J.A., Moussa M., Nery J.R., Nicovich P.R., Niu S.Y., Orvis J., Osteen J.K., Owen S., Palmer C.R., Pham T., Plongthongkum N., Poirion O., Reed N.M., Rimorin C., Rivkin A., Romanow W.J., Sedeño-Cortés A.E., Siletti K., Somasundaram S., Sulc J., Tieu M., Torkelson A., Tung H., Wang X., Xie F., Yanny A.M., Zhang R., Ament S.A., Behrens M.M., Bravo H.C., Chun J., Dobin A., Gillis J., Hertzano R., Hof P.R., Höllt T., Horwitz G.D., Keene C.D., Kharchenko P.V., Ko A.L., Lelieveldt B.P., Luo C., Mukamel E.A., Pinto-Duarte A., Preissl S., Regev A., Ren B., Scheuermann R.H., Smith K., Spain W.J., White O.R., Koch C., Hawrylycz M., Tasic B., Macosko E.Z., McCarroll S.A., Ting J.T., Zeng H., Zhang K., Feng G., Ecker J.R., Linnarsson S, & Lein E.S. (2021). Comparative cellular analysis of motor cortex in human, marmoset and mouse. Nature, 598(7879), 111-119.

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Postmortem Brain Tissue Preparation for RNA-Seq

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Whole postmortem brain specimens were bisected through the midline and individual hemispheres were embedded in alginate for slabbing. Coronal brain slabs were cut at 0.5-1cm intervals through each hemisphere and the slabs were frozen in a bath of dry ice and isopentane and stored at -80°C. For RNA-sequencing experiments, middle temporal gyrus (MTG) was identified on slabs of interest and removed for further sectioning. MTG tissue was then thawed in a buffer containing PBS supplemented with 10mM DL-Dithiothreitol (DTT, Sigma Aldrich), mounted on a vibratome (Leica), and sectioned at 500µm in the coronal plane. Sections were transferred to a fluorescent Nissl staining solution (Neurotrace 500/525, ThermoFisher Scientific) prepared in PBS with 10mM DTT and 0.5% RNasin Plus RNase inhibitor (Promega). After staining for 5 min, sections were visualized on a fluorescence dissecting microscope (Leica) and layer 1 was microdissected using a needle blade micro-knife (Fine Science Tools).

Boldog E., Bakken T.E., Hodge R.D., Novotny M., Aevermann B.D., Baka J., Bordé S., Close J.L., Diez-Fuertes F., Ding S.L., Faragó N., Kocsis Á.K., Kovács B., Maltzer Z., McCorrison J.M., Miller J.A., Molnár G., Oláh G., Ozsvár A., Rózsa M., Shehata S.I., Smith K.A., Sunkin S.M., Tran D.N., Venepally P., Wall A., Puskás L.G., Barzó P., Steemers F.J., Schork N.J., Scheuermann R.H., Lasken R.S., Lein E.S, & Tamás G. (2018). Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type. Nature neuroscience, 21(9), 1185-1195.

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Macaque Motor Cortex Transcriptional Profiling

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Tissue was obtained from three macaque animals (aged 3–17 years, male and female; Extended Data Table 2) as above. As described for humans, M1 was isolated from thawed slabs using fluorescent Nissl staining (Neurotrace 500/525, ThermoFisher Scientific). Stained sections were screened for histological hallmarks of primary motor cortex, and L5 was dissected. Nuclei were isolated from the dissected layer; gene expression was quantified with 10× Chromium v3 using the Mmul_10 genome annotation; nuclei that passed quality-control criteria were clustered; and a taxonomy of glutamatergic types was defined. To identify which clusters in our three-species taxonomy aligned with macaque clusters from our L5 dissected Cv3 dataset, we carried out an identical integration workflow on glutamatergic neurons to that used for the three-species integration. Macaque clusters were assigned subclass labels on the basis of their corresponding alignment with subclasses from the other species.

Bakken T.E., Jorstad N.L., Hu Q., Lake B.B., Tian W., Kalmbach B.E., Crow M., Hodge R.D., Krienen F.M., Sorensen S.A., Eggermont J., Yao Z., Aevermann B.D., Aldridge A.I., Bartlett A., Bertagnolli D., Casper T., Castanon R.G., Crichton K., Daigle T.L., Dalley R., Dee N., Dembrow N., Diep D., Ding S.L., Dong W., Fang R., Fischer S., Goldman M., Goldy J., Graybuck L.T., Herb B.R., Hou X., Kancherla J., Kroll M., Lathia K., van Lew B., Li Y.E., Liu C.S., Liu H., Lucero J.D., Mahurkar A., McMillen D., Miller J.A., Moussa M., Nery J.R., Nicovich P.R., Niu S.Y., Orvis J., Osteen J.K., Owen S., Palmer C.R., Pham T., Plongthongkum N., Poirion O., Reed N.M., Rimorin C., Rivkin A., Romanow W.J., Sedeño-Cortés A.E., Siletti K., Somasundaram S., Sulc J., Tieu M., Torkelson A., Tung H., Wang X., Xie F., Yanny A.M., Zhang R., Ament S.A., Behrens M.M., Bravo H.C., Chun J., Dobin A., Gillis J., Hertzano R., Hof P.R., Höllt T., Horwitz G.D., Keene C.D., Kharchenko P.V., Ko A.L., Lelieveldt B.P., Luo C., Mukamel E.A., Pinto-Duarte A., Preissl S., Regev A., Ren B., Scheuermann R.H., Smith K., Spain W.J., White O.R., Koch C., Hawrylycz M., Tasic B., Macosko E.Z., McCarroll S.A., Ting J.T., Zeng H., Zhang K., Feng G., Ecker J.R., Linnarsson S, & Lein E.S. (2021). Comparative cellular analysis of motor cortex in human, marmoset and mouse. Nature, 598(7879), 111-119.

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5

Postmortem Motor Cortex Tissue Isolation

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Male and female donors 18–68 years of age with no known history of neuropsychiatric or neurological conditions (‘control’ cases) were considered for inclusion in this study (Extended Data Table 1). Routine serological screening for infectious disease (HIV, hepatitis B and hepatitis C) was conducted using donor blood samples, and only those donors who were negative for all three tests were considered for inclusion. Only those specimens with RNA integrity (RIN) values of 7.0 or more were considered for inclusion. Postmortem brain specimens were processed as described^{3 (link)}. Briefly, coronal brain slabs were cut at 1 cm intervals and frozen for storage at −80 °C until further use. Putative hand and trunk-lower limb regions of the primary motor cortex were identified, removed from slabs of interest, and subdivided into smaller blocks. One block from each donor was processed for cryosectioning and fluorescent Nissl staining (Neurotrace 500/525, ThermoFisher Scientific). Stained sections were screened for histological hallmarks of primary motor cortex. After verifying that regions of interest contained M1, blocks were processed for nucleus isolation as described below.

Bakken T.E., Jorstad N.L., Hu Q., Lake B.B., Tian W., Kalmbach B.E., Crow M., Hodge R.D., Krienen F.M., Sorensen S.A., Eggermont J., Yao Z., Aevermann B.D., Aldridge A.I., Bartlett A., Bertagnolli D., Casper T., Castanon R.G., Crichton K., Daigle T.L., Dalley R., Dee N., Dembrow N., Diep D., Ding S.L., Dong W., Fang R., Fischer S., Goldman M., Goldy J., Graybuck L.T., Herb B.R., Hou X., Kancherla J., Kroll M., Lathia K., van Lew B., Li Y.E., Liu C.S., Liu H., Lucero J.D., Mahurkar A., McMillen D., Miller J.A., Moussa M., Nery J.R., Nicovich P.R., Niu S.Y., Orvis J., Osteen J.K., Owen S., Palmer C.R., Pham T., Plongthongkum N., Poirion O., Reed N.M., Rimorin C., Rivkin A., Romanow W.J., Sedeño-Cortés A.E., Siletti K., Somasundaram S., Sulc J., Tieu M., Torkelson A., Tung H., Wang X., Xie F., Yanny A.M., Zhang R., Ament S.A., Behrens M.M., Bravo H.C., Chun J., Dobin A., Gillis J., Hertzano R., Hof P.R., Höllt T., Horwitz G.D., Keene C.D., Kharchenko P.V., Ko A.L., Lelieveldt B.P., Luo C., Mukamel E.A., Pinto-Duarte A., Preissl S., Regev A., Ren B., Scheuermann R.H., Smith K., Spain W.J., White O.R., Koch C., Hawrylycz M., Tasic B., Macosko E.Z., McCarroll S.A., Ting J.T., Zeng H., Zhang K., Feng G., Ecker J.R., Linnarsson S, & Lein E.S. (2021). Comparative cellular analysis of motor cortex in human, marmoset and mouse. Nature, 598(7879), 111-119.

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Postmortem Brain Tissue Preparation for RNA-Seq

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Whole postmortem brain specimens were bisected through the midline and individual hemispheres were embedded in alginate for slabbing. Coronal brain slabs were cut at 0.5-1cm intervals through each hemisphere and the slabs were frozen in a bath of dry ice and isopentane and stored at -80°C. For RNA-sequencing experiments, middle temporal gyrus (MTG) was identified on slabs of interest and removed for further sectioning. MTG tissue was then thawed in a buffer containing PBS supplemented with 10mM DL-Dithiothreitol (DTT, Sigma Aldrich), mounted on a vibratome (Leica), and sectioned at 500µm in the coronal plane. Sections were transferred to a fluorescent Nissl staining solution (Neurotrace 500/525, ThermoFisher Scientific) prepared in PBS with 10mM DTT and 0.5% RNasin Plus RNase inhibitor (Promega). After staining for 5 min, sections were visualized on a fluorescence dissecting microscope (Leica) and layer 1 was microdissected using a needle blade micro-knife (Fine Science Tools).

Boldog E., Bakken T.E., Hodge R.D., Novotny M., Aevermann B.D., Baka J., Bordé S., Close J.L., Diez-Fuertes F., Ding S.L., Faragó N., Kocsis Á.K., Kovács B., Maltzer Z., McCorrison J.M., Miller J.A., Molnár G., Oláh G., Ozsvár A., Rózsa M., Shehata S.I., Smith K.A., Sunkin S.M., Tran D.N., Venepally P., Wall A., Puskás L.G., Barzó P., Steemers F.J., Schork N.J., Scheuermann R.H., Lasken R.S., Lein E.S, & Tamás G. (2018). Transcriptomic and morphophysiological evidence for a specialized human cortical GABAergic cell type. Nature neuroscience, 21(9), 1185-1195.

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Postmortem Brain Tissue Sectioning

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Whole postmortem brain specimens were transported to the Allen Institute on ice. Standard processing of whole brain specimens involved bisecting the brain through the midline and embedding of individual hemispheres in Cavex Impressional Alginate for slabbing. Coronal brain slabs were cut at 1cm intervals through each hemisphere and individual slabs were frozen in a slurry of dry ice and isopentane. Slabs were then vacuum sealed and stored at −80°C until the time of further use.
Middle temporal gyrus (MTG) was identified on and removed from frozen slabs of interest, and subdivided into smaller blocks for further sectioning. Individual tissue blocks were processed by thawing in PBS supplemented with 10mM DL-Dithiothreitol (DTT, Sigma Aldrich), mounting on a vibratome (Leica), and sectioning at 500μm in the coronal plane. Sections were placed in fluorescent Nissl staining solution (Neurotrace 500/525, ThermoFisher Scientific) prepared in PBS with 10mM DTT and 0.5% RNasin Plus RNase inhibitor (Promega) and stained for 5 min on ice. After staining, sections were visualized on a fluorescence dissecting microscope (Leica) and cortical layers were individually microdissected using a needle blade micro-knife (Fine Science Tools).

Hodge R.D., Bakken T.E., Miller J.A., Smith K.A., Barkan E.R., Graybuck L.T., Close J.L., Long B., Johansen N., Penn O., Yao Z., Eggermont J., Hollt T., Levi B.P., Shehata S.I., Aevermann B., Beller A., Bertagnolli D., Brouner K., Casper T., Cobbs C., Dalley R., Dee N., Ding S.L., Ellenbogen R.G., Fong O., Garren E., Goldy J., Gwinn R.P., Hirschstein D., Keene C.D., Keshk M., Ko A.L., Lathia K., Mahfouz A., Maltzer Z., McGraw M., Nguyen T.N., Nyhus J., Ojemann J.G., Oldre A., Parry S., Reynolds S., Rimorin C., Shapovalova N.V., Somasundaram S., Szafer A., Thomsen E.R., Tieu M., Quon G., Scheuermann R.H., Yuste R., Sunkin S.M., Lelieveldt B., Feng D., Ng L., Bernard A., Hawrylycz M., Phillips J.W., Tasic B., Zeng H., Jones A.R., Koch C, & Lein E.S. (2019). Conserved cell types with divergent features in human versus mouse cortex. Nature, 573(7772), 61-68.

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Histological Verification of Fiber-Optic Implants

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Mice were decapitated, and heads were transferred into 4% phosphate-buffered formaldehyde (PFA). After at least 10 days, fiber-optic cannulas were removed, brains were dissected and transferred into 4% phosphate-buffered PFA containing 25% sucrose. After equilibration in this sucrose solution, brains were cut at 30 µm on a freezing microtome, stained with NeuroTrace™500/525 (1:250; ThermoFisher Scientific) and imaged for determination of fiber-optic cannula placements.

Engström Ruud L., Pereira M.M., de Solis A.J., Fenselau H, & Brüning J.C. (2020). NPY mediates the rapid feeding and glucose metabolism regulatory functions of AgRP neurons. Nature Communications, 11, 442.

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Marmoset Brain Nuclei Isolation and Sequencing

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Marmoset experiments were approved by and in accordance with Massachusetts Institute of Technology IACUC protocol number 051705020. Two adult marmosets (2.3 and 3.1 years old; one male, one female; Extended Data Table 2) were deeply sedated by intramuscular injection of ketamine (20-40 mg/kg) or alfaxalone (5-10 mg/kg), followed by intravenous injection of sodium pentobarbital (10-30 mg/kg). When pedal withdrawal reflex was eliminated and/or respiratory rate was diminished, animals were transcardially perfused with ice-cold sucrose-HEPES buffer. Whole brains were rapidly extracted into fresh buffer on ice. Sixteen 2-mm coronal blocking cuts were rapidly made using a custom-designed marmoset brain matrix. Coronal slabs were snap-frozen in liquid nitrogen and stored at -80°C until use.
As with human samples, M1 was isolated from thawed slabs using fluorescent Nissl staining (Neurotrace 500/525, ThermoFisher Scientific). Stained sections were screened for histological hallmarks of primary motor cortex. Nuclei were isolated from the dissected regions following this protocol: https://www.protocols.io/view/extraction-of-nuclei-from-brain-tissue-2srged6 and processed using the 10x Chromium Single-Cell 3' Reagent Kit v3. 10x chip loading and sample processing was done according to the manufacturer's protocol.

Bakken T.E., Jorstad N.L., Hu Q., Lake B.B., Tian W., Kalmbach B., Crow M., Hodge R.D., Krienen F.M., Sorensen S.A., Eggermont J., Yao Z., Aevermann B.D., Aldridge A., Bartlett A., Bertagnolli D., Casper T., Castanon R., Crichton K., Daigle T.L., Dalley R., Dee N., Dembrow N., Diep D., Ding S., Dong W., Fang R., Fischer S., Goldman M., Goldy J., Graybuck L.T., Herb B.R., Hou X., Kancherla J., Kroll M., Lathia K., Lew B.v., Li Y.E., Liu C.S., Liu H., Lucero J., Mahurkar A., McMillen D., Miller J.A., Moussa M., Nery J.R., Nicovich P.R., Orvis J., Osteen J.K., Owen S.F., Palmer C.R., Pham T., Plongthongkum N., Poirion O., Reed N., Rimorin C., Rivkin A., Romanow W.J., Sedeño-Cortés A.E., Siletti K., Somasundaram S., Šulc J., Tieu M., Torkelson A., Tung H., Wang X., Xie F., Yanny A.M., Zhang Y., Ament S.A., Behrens M.M., Bravo H.C., Chun J., Dobin A., Gillis J., Hertzano R., Hof P.R., Höllt T., Horwitz G.D., Keene C.D., Kharchenko P.V., Ko A.L., Lelieveldt B.P.F., Luo C., Mukamel E.A., Preißl S., Regev A., Ren B., Scheuermann R.H., Smith K.A., Spain W.J., White O., Koch C., Hawrylycz M., Tasic B., Macosko E.Z., McCarroll S.A., Ting J.T., Zeng H., & Zhang K. (2020). Evolution of cellular diversity in primary motor cortex of human, marmoset monkey, and mouse.

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10

Postmortem motor cortex isolation

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Male and female donors 18-68 years of age with no known history of neuropsychiatric or neurological conditions ('control' cases) were considered for inclusion in the study (Extended Data Table 1). Routine serological screening for infectious disease (HIV, Hepatitis B, and Hepatitis C) was conducted using donor blood samples and only donors negative for all three tests were considered for inclusion in the study. Only specimens with RNA integrity (RIN) values ≥7.0 were considered for inclusion in the study.
Postmortem brain specimens were processed as previously described 2 . Briefly, coronal brain slabs were cut at 1cm intervals and frozen for storage at -80°C until the time of further use. Putative hand and trunk-lower limb regions of the primary motor cortex were identified, removed from slabs of interest, and subdivided into smaller blocks. One block from each donor was processed for cryosectioning and fluorescent Nissl staining (Neurotrace 500/525, ThermoFisher Scientific). Stained sections were screened for histological hallmarks of primary motor cortex. After verifying that regions of interest contained M1, blocks were processed for nucleus isolation as described below.

Bakken T.E., Jorstad N.L., Hu Q., Lake B.B., Tian W., Kalmbach B., Crow M., Hodge R.D., Krienen F.M., Sorensen S.A., Eggermont J., Yao Z., Aevermann B.D., Aldridge A., Bartlett A., Bertagnolli D., Casper T., Castanon R., Crichton K., Daigle T.L., Dalley R., Dee N., Dembrow N., Diep D., Ding S., Dong W., Fang R., Fischer S., Goldman M., Goldy J., Graybuck L.T., Herb B.R., Hou X., Kancherla J., Kroll M., Lathia K., Lew B.v., Li Y.E., Liu C.S., Liu H., Lucero J., Mahurkar A., McMillen D., Miller J.A., Moussa M., Nery J.R., Nicovich P.R., Orvis J., Osteen J.K., Owen S.F., Palmer C.R., Pham T., Plongthongkum N., Poirion O., Reed N., Rimorin C., Rivkin A., Romanow W.J., Sedeño-Cortés A.E., Siletti K., Somasundaram S., Šulc J., Tieu M., Torkelson A., Tung H., Wang X., Xie F., Yanny A.M., Zhang Y., Ament S.A., Behrens M.M., Bravo H.C., Chun J., Dobin A., Gillis J., Hertzano R., Hof P.R., Höllt T., Horwitz G.D., Keene C.D., Kharchenko P.V., Ko A.L., Lelieveldt B.P.F., Luo C., Mukamel E.A., Preißl S., Regev A., Ren B., Scheuermann R.H., Smith K.A., Spain W.J., White O., Koch C., Hawrylycz M., Tasic B., Macosko E.Z., McCarroll S.A., Ting J.T., Zeng H., & Zhang K. (2020). Evolution of cellular diversity in primary motor cortex of human, marmoset monkey, and mouse.

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Neurotrace 500 525

Lab products found in correlation

14 protocols using neurotrace 500 525

Postmortem Brain Tissue Sectioning

Transcardial Perfusion and Nuclei Isolation for Marmoset M1 Cortex

Postmortem Brain Tissue Preparation for RNA-Seq

Macaque Motor Cortex Transcriptional Profiling

Postmortem Motor Cortex Tissue Isolation

Postmortem Brain Tissue Preparation for RNA-Seq

Postmortem Brain Tissue Sectioning

Histological Verification of Fiber-Optic Implants

Marmoset Brain Nuclei Isolation and Sequencing

Postmortem motor cortex isolation

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