GSM3097626: L3_I2_BAS_B12; Homo sapiens; RNA-Seq
Viable organoids were thawed and washed using DMEM, and digested with 0.05% trypsin (Corning 25-052-CI) containing DNase (Sigma Aldrich D4263-5VL) to generate single cell suspension. Cells were stained for FACS using fluorescently labeled antibodies for CD31 (eBiosciences 48-0319-42), CD45 (eBiosciences 48-9459-42), EpCAM (eBiosciences 50-9326-42), CD49f (eBiosciences 12-0495-82), SytoxBlue (Life Technologies S34857). We only proceeded with samples showing at least 80% viability as measured using SytoxBlue in FACS Sorted cells were washed and resuspended at a concentration of approximately 500 cells/µl. For microfluidics-enabled scRNAseq, cell suspensions were mixed with Fluidigm C1 Suspension Reagents (Fluidigm 100-5315) at a ratio of 8:2 before loading mix onto C1 chip (Fluidigm 100-5760). Bright field images of captured cells were collected using a Keyence BZ-X710 microscope (Keyence Corporation, Itasca, Illinois, USA). Single-cell RNA isolation and amplification were performed using the Fluidigm C1 Single Cell Auto Prep IFC following the Fluidigm Protocol: 100-7168 I1. RNA spike-in controls were omitted. cDNA library preparation were performed following the Fluidigm C1 Protocol: 100-7168 I1. Reagents Kits v2 User Guide: CG00052 Rev B.     Quantification of cDNA libraries was performed using Qubit dsDNA HS Assay Kit (Life Technologies Q32851) and high-sensitivity DNA chips (Agilent. 5067-4626). Quantification of library construction was performed using KAPA qPCR (Kapa Biosystems KK4824). For microfluidics-enabled scRNAseq libraries, we generally multiplexed 96 cells per lane on an Illumina HiSeq2500 resulting in a calculated depth of ~1.6 million reads per cell (Illumina Rapid PE kit v2 402-4002 and Rapid SBS kit v2 FC 401-4022).
GEO Accession
GSM3097626