Illumina HiSeq 4000 paired end sequencing; Single-cell transcriptome profiling of Sox2-low iPS cells
Single-cell transcriptome profiling of Sox2-low iPS cells
Single cells were deposited by flow cytometry (BD Influx 5) into individual wells of a 96-well PCR plate containing lysis buffer. Neural stem (NS) cells were cultured in DMEM/F-12 (Thermo Fisher Scientific), 1:100 v/v B27 supplement (Thermo Fisher Scientific), 1:200 v/v N2 supplement (University of Cambridge), 4.5μM HEPES (Thermo Fisher Scientific), 1x non-essential amino acids, 0.1mM 2-mercaptoethanol, 1x penicillin-streptomycin, 0.03M glucose and 120μg/ml BSA (Thermo Fisher Scientific) supplemented with 10ng/ml EGF (Peprotech) and 20ng/ml FGF2 (University of Cambridge).  Induced pluripotent stem (iPS) cells were cultured in KSR basal medium (GMEM basal medium with 10% knockout serum replacement, 1% fetal calf serum) or N2B27 basal medium (DMEM/F-12 and Neurobasal (Thermo Fisher Scientific) in a 1:1 ratio, 1:200 v/v N2 and 1:100 v/v B27 supplement, 2mM L-glutamine, 1x penicillin-streptomycin, 0.1mM 2-mercaptoethanol) with 20ng/ml mouse LIF, 3μM CHIR99021 and 1μM PD0325901 (Stewart lab, Biotechnology Center TU Dresden). Platinum-E (HEK293T) cells were transfected with 9μg of the following plasmids: pMXs c-Myc, pMXs Oct4, pMXs Sox2, and pMXs Klf4 using FuGENE 6 (Roche) for retroviral production. Supernatants were collected after 48 hours, combined, mixed with 4μg/ml polybrene (Sigma-Aldrich) and passed through a 0.45 μm cellulose acetate filter. NS cells were exposed to this solution for 24 hours, after which they were returned to NS medium for 2 days before being placed in serum/LIF medium (GMEM (Sigma-Aldrich), 1x non-essential amino acids (Gibco), 1x penicillin-streptomycin (Sigma-Aldrich), 1mM sodium pyruvate (Sigma-Aldrich), 0.1mM 2-mercaptoethanol (Thermo Fisher Scientific) and 2mM L-glutamine (Thermo Fisher Scientific)) with 10% fetal calf serum (Labtech) and 20ng/ml mouse LIF (University of Cambridge)) to form reprogramming intermediates (pre-iPS cells). Cells were then cultured in KSR/2i/LIF medium for iPS cell colony formation. Total RNA was isolated using the RNeasy Mini kit (Qiagen). Single-cell RNA-seq was performed with the SmartSeq2 protocol (Picelli et al., Nat Protoc. 9:171-81). Sequencing libraries were prepared with the Illumina Nextera XT DNA kit.