title: NextSeq 500 sequencing; Transcriptomic profile of human induced pluripotent stem cells at single cell resolution.
design: Transcriptomic profile of human induced pluripotent stem cells at single cell resolution.
library_construction_protocol: Human skin biopsies were obtained from subjects over the age of 18 years. Fibroblasts were cultured in DMEM with high glucose, 10% fetal bovine serum (FBS), L-glutamine, 100 U/mL Penicillin and 100 µg/mL streptomycin (all from Thermo Fisher Scientific, USA). All cell lines were mycoplasma-free (MycoAlert mycoplasma detection kit, Lonza, Switzerland). Fibroblasts at passage (p) 8-10 were used for reprogramming. A TECAN liquid handling platform was used to cultivate cells, as described in (Crombie et al., 2017, SLAD Discov.). iPSCs were generated by nucleofection (AmaxaTM NucleofectorTM) with episomal vectors expressing OCT4, SOX2, KLF4, L-MYC, LIN28 and shRNA against p53 (Okita et al., 2011, Nat Methods) in feeder- and serum-free conditions using TeSRTM-E7TM medium (Stem Cell Technologies, Canada) as described previously (Crombie et al., 2017, SLAD Discov.). The reprogrammed cells were maintained on the automated platform using TeSRTM-E7TM medium, with daily medium change. Pluripotent cells were selected by sorting anti-human TRA-1-60 Microbeads using a MultiMACS (Miltenyi, Germany) as described in (Crombie et al., 2017, SLAD Discov.). Cell number was determined and cell were subsequently plated onto vitronectin XFTM (Stem Cell Technologies) in TeSRTM-E8TM medium (Stem Cell Technologies) or StemFlexTM medium (Thermo Fisher Scientific). Subsequent culturing was performed on the automated platform using TeSRTM-E8TM or StemFlexTM medium, changing medium every two days. Passaging was performed weekly on the automated platform using ReLeSRTM (Stem Cell Technologies) onto vitronectin XFTM coated plates as described in (Crombie et al., 2017, SLAD Discov.) Cells were harvested using ReleSRTM, colonies were dissociated into single cell suspension. Cells were counted and assessed for viability with Trypan Blue using a Countess II automated counter (Thermo Fisher Scientific), then pooled at a concentration of 391-663 cells/µL (3.91x10^5-6.63x10^5 cells/mL). Final cell viability estimates ranged between 85-97%. Cells were partitioned and barcoded using high-throughput droplet 10X Genomics ChromiumTM Controller (10X Genomics, USA) and the Single Cell 3' Library and Gel Bead Kit (V2; 10X Genomics; PN-120237). GEM generation and barcoding, cDNA amplification, and library construction were performed according to standard protocol.  Library construction were performed according to 10X standard protocol.  
attributes.tag: Experimental Factor: replicate
attributes.value: 1