title: GSM4138489: WT_undiff_scRNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: H9 hESCs and differentiated hESCs were dissociated by Corning™ 0.25% Trypsin (Corning™ 25053CI) to single cells. Single cell 3' RNA-seq experiments were conducted using the Chromium single cell system (10x Genomics, Inc) and Illumina sequencers at the Center of Molecular Genetics (CMG) of Indiana University School of Medicine. Cell suspension was first inspected on the Countess II FL (Thermo Fisher Scientific) and under microscope for cell number, cell viability, and cell size. Depending on the quality of the initial cell suspension, the single cell preparation included centrifugation, re-suspension, and filtration to remove cell debris, dead cells and cell aggregates. Single cell capture and library preparation were carried out according to the Chromium Single cell 3' Reagent kits V2 User Guide (10X Genomics PN-120267, PN-1000009, PN-120262). Appropriate number of cells were loaded on a multiple-channel micro-fluidics chip of the Chromium Single Cell Instrument (10x Genomics) with a targeted cell recovery of 10,000. Single cell gel beads in emulsion containing barcoded oligonucleotides and reverse transcriptase reagents were generated with the v2 single cell reagent kit (10X Genomics). Following cell capture and cell lysis, cDNA was synthesized and amplified. Illumina sequencing libraries were then prepared with the amplified cDNA. The resulting libraries were assessed with an Agilent TapeStation or Bioanalyzer 2100. The final libraries of the undifferentiated samples were sequenced using a custom program on Illumina NextSeq 500/550; and the libraries of the differentiated samples were sequenced on Illumina NovaSeq 6000. 26 bp of cell barcode and UMI sequences, and 91 or 98 bp RNA reads were generated with Illumina NextSeq500/550 or NovaSeq 6000. Single cell 3' RNA-seq experiments were conducted using the Chromium single cell system (10x Genomics, Inc) and Illumina sequencers at the Center of Molecular Genetics (CMG) of Indiana University School of Medicine. Cell suspension was first inspected on the Countess II FL (Thermo Fisher Scientific) and under microscope for cell number, cell viability, and cell size. Depending on the quality of the initial cell suspension, the single cell preparation included centrifugation, re-suspension, and filtration to remove cell debris, dead cells and cell aggregates. Single cell capture and library preparation were carried out according to the Chromium Single cell 3' Reagent kits V2 User Guide (10X Genomics PN-120267, PN-1000009, PN-120262). Appropriate number of cells were loaded on a multiple-channel micro-fluidics chip of the Chromium Single Cell Instrument (10x Genomics) with a targeted cell recovery of 10,000. Single cell gel beads in emulsion containing barcoded oligonucleotides and reverse transcriptase reagents were generated with the v2 single cell reagent kit (10X Genomics). Following cell capture and cell lysis, cDNA was synthesized and amplified. Illumina sequencing libraries were then prepared with the amplified cDNA. The resulting libraries were assessed with an Agilent TapeStation or Bioanalyzer 2100.
attributes.tag: GEO Accession
attributes.value: GSM4138489