GSM2824789: BJ_MYO_R2_T24_B12_scRNA-seq; Homo sapiens; RNA-Seq
To​ ​perform​ ​reprogramming​ ​and​ ​differentiation​ ​experiments​ ​HSMM​ ​and​ ​hFib-MYOD​ ​cells​ ​were plated​ ​in​ ​24-well​ ​formats​ ​at​ ​a​ ​density​ ​of​ ​50.000​ ​cells​ ​per​ ​well.​ ​Gelatin​ ​was​ ​sometime​ ​used​ ​as​ ​a coating​ ​agent​ ​with​ ​no​ ​significant​ ​difference​ ​with​ ​respect​ ​to​ ​uncoated​ ​dishes.​ ​Differentiation​ ​and reprogramming​ ​was​ ​induced​ ​using​ ​a​ ​differentiation​ ​media​ ​containing​ ​alpha-mem​ ​supplemented with​ ​glutamax​ ​and​ ​2%​ ​HS​ ​(ThermoFisher​ ​Scientific),​ ​supplemented​ ​with​ ​2ug/ml​ ​of​ ​doxycycline to​ ​enact​ ​MYOD​ ​expression.​ ​When​ ​indicated,​ ​Insulin​ ​was​ ​used​ ​at​ ​8ug/ml​ ​(ThermoFisher Scientific),​ ​BMP​ ​inhibitor​ ​LDN-193189​ ​was​ ​used​ ​at​ ​0.1uM​ ​(Stemgent),​ ​LSD1​ ​inhibitor​ ​RN-1​ ​was used​ ​at​ ​1uM​ ​(EMD​ ​Millipore). At​ ​the​ ​indicated​ ​time​ ​points,​ ​cells​ ​were​ ​harvested​ ​by​ ​gentle​ ​dissociation​ ​using​ ​TrypLE (ThermoFisher​ ​Scientific)​ ​and​ ​processed​ ​for​ ​full-length​ ​transcriptome​ ​sequencing​ ​using​ ​the Fluidigm​ ​C1​ ​Single​ ​Cell​ ​or​ ​bulk​ ​mRNA​ ​sequencing​ ​as​ ​previously​ ​described​ (​​ Trapnell​ ​et​ ​al., 2014)​.​ ​Cells​ ​were​ ​loaded​ ​onto​ ​the​ ​microfluidic​ ​chip​ ​at​ ​a​ ​concentration​ ​of​ ​~250​ ​cells/μl​ ​to maximize​ ​the​ ​number​ ​of​ ​single​ ​cells​ ​captured​ ​and​ ​minimize​ ​the​ ​occurrence​ ​of​ ​doublets.​ ​​ ​In some​ ​instances,​ ​cells​ ​were​ ​reloaded​ ​if​ ​the​ ​majority​ ​of​ ​capture​ ​sites​ ​were​ ​not​ ​occupied​ ​on​ ​the first​ ​attempt.​ ​As​ ​in​ ​Trapnell​ ​et​ ​al,​ ​2014,​ ​captured​ ​cells​ ​were​ ​scored​ ​by​ ​manual​ ​on-chip microscopic​ ​inspection​ ​to​ ​determine​ ​if​ ​they​ ​were​ ​singletons​ ​and​ ​free​ ​of​ ​other​ ​debris.
GEO Accession
GSM2824789