alias: GSM2314269
title: GSM2314269: 54Dn1b_C06; Homo sapiens; RNA-Seq
library_construction_protocol: 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. CelSeq protocol with a few modifications. Single cells were sorted with a FACSAria (BD) into 96-well plates containing 1.2 μL 2× CellsDirect Buffer (Thermo Fisher) with 0.1 μL of External RNA Controls Consortium (ERCC) control RNAs diluted to 1 × 10-6 molecules (Thermo Fisher). After sorting, plates were then frozen and stored at -80C. For library preparation, plates were thawed on ice. mRNA was reverse transcribed using 1.25 pmol or 0.15625 pmol of oligoT  primer  carrying  a  cell-specific  8  NT  barcode  and  a  5  NT  unique  molecular  identifier  (UMI)  (Islam  et  al., 2014) (see Table S4). Barcode design ensured at least three nucleotide differences from any other barcode. Samples were  incubated  in a  PCR  machine  (Tetrad,  BioRad)  at  70  °C  with a  70 °C  heated  lid  for  3  min,  spun,  and heated again for two more minutes. Samples were reverse transcribed using Superscript III (Thermo Fisher) for two hours at 50 °C with a 52 °C lid and subsequently treated with 1 μL of ExoSAP-IT (Affymetrix). Samples were cooled on ice  for  second  strand  synthesis,  where  Second  Strand  Synthesis  Buffer,  dNTPs,  DNA  Polymerase,  and  RNAse  H (NEB) were added to the samples for a 10 μL total volume and incubated at 16 °C for 2 h. Single cells were pooled by 24 wells per library, with each library containing a water-only well and an ERCC-only well. Single cell pools or population  RNA  libraries  were  purified  with  an  equal  volume  of RNA  Clean  Beads  (Beckman  Coulter),  linearly amplified at 37 C for 15 h using the HiScribe T7 High Yield RNA Synthesis kit (NEB), and treated with DNase I (Thermo Fisher). RNA  was fragmented using the NEBNext RNA Fragmentation Module (NEB), purified using an equal volume of RNA Clean Beads, and visualized (RNA Pico Kit, Bioanalyzer 2100, Agilent). The RNA fragments were repaired by treating with Antarctic Phosphatase and Polynucleotide Kinase (NEB)  and purified with an equal volume of  RNA Clean Beads. cDNA libraries were made using the NEBNext Small Library Prep Kit according to the  manufacturer’s  protocol,  except  Superscript  III  was  used  for  the  RT  step. Index  primers  were  used  in  PCR amplification.  Libraries  were  purified  using  an  equal  volume  of  RNA  Clean  Beads  and  were  quantified  on  the Bioanalyzer using the DNA High Sensitivity Kit (Agilent). Approximately 160-200 nmol of a pool of libraries were size  selected  to  exclude  species  <180  bp  on  a  2%  Dye-Free  cassette  on  the  Pippin  Prep  (Sage)  and  Speed  Vac concentrated    to    approximately    14    μL.    Libraries    were    then quantified    by    qRT-PCR    using    p5    (5’-AATGATACGGCGACCACCGAGA-3’) and  p7   (5’-CAAGCAGAAGACGGCATACGAGAT-3’)   primers   and visualized (DNA High Sensitivity Kit, Bioanalyzer 2100). Library pools were then sequenced on an Illumina HiSeq  using a custom read1 primer (5’-TCTACACGTTCAGAGTTCTACAGTCCGACGATC-3’) and the Illumina primer HP10. Standard Illumina primers HP12 and HP11 were used for the index read and the transcript read, respectively. PE50 kits (Illumina) were used for sequencing with read lengths of 25 nt, 6 nt, and 47 nt for read1, index, and read2, respectively.
identifiers.namespace: GEO
identifiers.id: GSM2314269