Single-cell Transcriptome Profiling with Fluidigm and Smart-seq2

Cells were dissociated using accutase supplemented with DNase (Worthington Biochemical, LK003170). A 100-μm nozzle was used for sorting. Cells were sorted by BD Facs Aria, 20 psi, 40–100 ev/s into 96-well plates (one cell per well) containing 5 μL lysis buffer. For Fluidigm Biomark, cells were single-cell sorted into 5 μL of 10 mM Tris (pH 8.0), 0.1 mM EDTA supplemented with SUPERase•In (0.1 U/μL final, Ambion, AM2696), and 0.5% NP40 (Thermo Scientific, PI-28324). For Smart-seq2 library prep, cells were single-cell sorted into 5 μL 1×TCL buffer (QIAGEN, 1031576). After sorting, plates were immediately sealed, spun down for 5 min at 550 × g, and flash frozen on dry ice. Sorted cells were then stored at −80°C. Details on single-cell expression profiling and transcriptomics analysis are provided in the Supplemental Experimental Procedures. To compare the piN cells to publicly available single-cell reference datasets (Pollen et al., 2014 (link), Darmanis et al., 2015 (link)), we performed integrated analysis using Seurat version (v.)2.1

Free full text: Click here

Nehme R., Zuccaro E., Dia Ghosh S., Li C., Sherwood J.L., Pietilainen O., Barrett L.E., Limone F., Worringer K.A., Kommineni S., Zang Y., Cacchiarelli D., Meissner A., Adolfsson R., Haggarty S., Madison J., Muller M., Arlotta P., Fu Z., Feng G, & Eggan K. (2018). Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission. Cell reports, 23(8), 2509-2523.

Publication 2018

LK003170 Facs Aria AM2696 PI-28324 1×TCL buffer

Accutase Aria Buffer Dnase Dry ice Edta Frozen Library Pollen Transcriptomics analysis Tris

Corresponding Organization : Harvard University

Other organizations : Novartis (United States), Umeå University, Center for Human Genetics, Massachusetts General Hospital, Novartis (Switzerland), McGovern Institute for Brain Research

Top 5 similar protocols

Variable analysis

independent variables

Cell dissociation using accutase supplemented with DNase
Cell sorting using BD Facs Aria with a 100-μm nozzle, 20 psi, 40–100 ev/s

dependent variables

Single-cell expression profiling and transcriptomics analysis

control variables

Cells were sorted into 96-well plates (one cell per well) containing 5 μL lysis buffer or 5 μL of 10 mM Tris (pH 8.0), 0.1 mM EDTA supplemented with SUPERase•In (0.1 U/μL final) and 0.5% NP40, or 5 μL 1×TCL buffer
Sorted cells were immediately sealed, spun down for 5 min at 550 × g, and flash frozen on dry ice before storage at −80°C

controls

No positive or negative controls were explicitly mentioned in the provided information.

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!