GSM2462675: 100PS_+_D100_y_3; Homo sapiens; RNA-Seq
To generate single cell suspensions, hESC-derived cultures were dissociated from plates using Accutase  (ThermoFisher)  at  37°C.    Light  trituration  using  a P1000  pipette  was  done  every  5  min  until  nearly  all clumps  had  been  dissociated  (up  to  1  h).    Cell  suspension  was  washed  and  filtered  through  a  40  μm  cell  strainer.  Cells were washed in PBS with 1% FBS and stained with 0.5-1 μg/mL DAPI.  Single-cell suspensions were loaded onto  a  FACSAria  II  SORP  (Becton  Dickinson)  and  sorted  directly  into  PCR  strip  tubes  or  plates  held  in  chilled aluminum  blocks.    Doublets  and  dead  cells  were  excluded  based  on  forward  scatter,  side  scatter  and  DAPI fluorescence. Sorting was done using the 130  μm nozzle with the sort mode set to single cell. Accuracy of single-cell  sorts  was  confirmed  by  sorting  DAPI-stained  fixed  cells  onto  a  dry  well  of  a  96-well  plate  and  analyzing  by fluorescence microscopy. Cells were sorted into 10K cell Citrine-negative (progenitors) or Citrine-positive (neurons) populations at each timepoint. These 10K cell subpopulations were processed for RNAseq via the SmartSeq2 kit. SmartSeq2 protocol. After  reverse  transcription  and  template  switching,  we amplified  cDNA  with  KAPA  HotStart  HIFI  2×  ReadyMix  (Kapa  Biosystems)  for  22  cycles  for  RNA  from  single primary cortical cells. We purified PCR products using Ampure XP beads (Beckman Coulter). We quantified cDNA using a High Sensitivity DNA Chip (Agilent) on a Bioanalyzer 2100 or with the Quant-iT PicoGreen dsDNA Assay   Kit  (Thermo  Fisher)  on  an  Enspire  plate  reader  (PerkinElmer).  We  used  1  ng  of  cDNA  to  generate  RNA-Seq libraries using the Nextera XT library prep system (Illumina). We carried out sequencing of human cortical cells the on Illumina HiSeq using 50 base paired-end reads
GEO Accession
GSM2462675