title: EV_24_2iLIF_Dox
organism: Mus musculus
description: Protocols: Cells were dissociated with Accutase (Millipore). ESCs and iPSCs were cultured in N2B27 medium supplemented with MEK and GSK3 inhibitors (2i) and leukaemia inhibitory factor (2iLIF). EpiSCs were cultured in N2B27 supplemented with FGF2, ActivinA and XAV939 (FAX). N2B27 medium comprised 1:1 DMEM/F-12 and Neurobasal (Gibco), 2 mM L-glutamine (Gibco), 1x penicillin-streptomycin (Sigma), 0.1 mM 2-mercaptoethanol (Gibco), 1% B27 (Gibco) and 0.5% N2 (homemade). As required, N2B27 was supplemented with 20 ng/ml murine LIF (Hyvonen lab, Cambridge), 3 μM CHIR99021 and 1 μM PD0325901 (Stewart lab, Dresden), 12.5 ng/ml FGF2 and 20 ng/ml ActivinA (Hyvonen lab, Cambridge), 6.25 µg/ml XAV939 (Tocris), 3 µM DMH2 (Tocris), or 0.6 µM LDN193189 (Sigma). For ESCs and iPSCs, culture vessels were coated with 0.15% gelatin (Sigma) in PBS (Sigma) and incubated in 7% CO2. For EpiSC culture and reprogramming experiments, tissue-culture flasks were coated with 10 µg/ml fibronectin (Millipore) in PBS (Sigma) and incubated in 7% CO2 and 5% O2. ESCs, iPSCs and EpiSCs were dissociated with accutase (Millipore) during passaging. For selected experiments, ESCs were cultured in serum+LIF medium containing GMEM (Sigma), 10% fetal bovine serum (Gibco), 1x non-essential amino acids (Gibco), 1 mM sodium pyruvate (Sigma), 2 mM L-glutamine (Gibco), 1x penicillin-streptomycin (Sigma), 0.1 mM 2-mercaptoethanol (Gibco). 20 ng/ml murine LIF (Hyvonen lab, Cambridge) and 10ng/ml BMP4 (Miltenyi Biotec) was supplemented as indicated. EpiSCs were plated in FA without selection at a density of 2000 cells/24-well or equivalent. For siRNA experiments, 10000/24-well or equivalent was used instead to compensate for transfection toxicity. The following day, reprogramming was induced by medium change to 2iLIF or subset components thereof as indicated. GY118F transgenic receptor was stimulated with 30 ng/ml human GCSF (Peprotech), whereas expression of other transgenes was induced with 1 µg/ml doxycycline (MP Biomedicals). After 4 days, transgene induction was withdrawn and 20 μg/ml blasticidin (Gibco) was applied to select for Rex1::dGFP.IRES.bsd reporter activity. Total RNA was isolated using the RNeasy protocol (Qiagen), including on-column DNaseI digestion. Sequencing libraries were prepared according to the SmartSeq2 protocol (Picelli et al., Nat Protoc. 9:171-81) with the following amendments: purified RNA was used and diluted to 5 ng/µl; ERCC spike-in controls (Invitrogen) were added at 1 µl of 1:10000 dilution per 5 ng; 13 cycles of amplification were used to obtain cDNA (rather than 21 used for single cells). Nextera XT reactions were scaled-down by half, using 0.4ng cDNA input per reaction.