Reprogramming experiments with B cells were performed as previously described (Di Stefano et al., 2014); with MEFs, macrophages and NSCs were conducted by plating 100.000 cells/well on gelatinized plates seeded with irradiated MEFs, using ESC medium supplemented with 2 µg/ml of doxycycline. For the isolation of iPSC lines, doxycycline was washed out after 15 days of reprogramming and colonies with ESC-like morphology were picked at 20 days before further passaging. iPSC lines were expanded for an additional 9 days (3 passages) to obtain P3 iPS cell lines or for 20 passages to obtain P20 iPS cell lines.
Growth protocol
ESCs and iPSCs were cultured on Mitomycin-C treated MEFs in KO-DMEM (Invitrogen) supplemented with 1% nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), 1,000 U/ml LIF (Millipore) and 15% fetal bovine serum (FBS, Invitrogen) (ESC medium). Naïve ESCs were cultured in serum-free N2B27 medium on gelatin-coated dishes. N2B27 medium (500ml) was generated by inclusion of the following: 240ml DMEM/F12 (Invitrogen), 240ml Neurobasal (Invitrogen), 5ml N2 supplement (Invitrogen), 10ml B27 supplement (Invitrogen), 1,000 U/ml LIF (Millipore), 1% nonessential amino acids (Invitrogen), 0.1mM β-mercaptoethanol (Invitrogen), and the small molecules PD0325901 (Stemgent, 1μM) and CHIR (Stemgent, 3μM). CD19+ pre-B cells and Mac1+ macrophages were isolated from bone marrow with monoclonal antibodies to CD19 and Mac-1 (BD Pharmingen) respectively, using MACS sorting (Miltenyi Biotech). B cells were grown in RPMI medium with 10% FBS and 10ng/ml IL-7 (Peprotech); macrophages in DMEM with 10% FBS and 10ng/ml each of CSF1 and IL-3 (Peprotech). MEFs were established from day 13.5 mouse embryos and cultured in DMEM containing 10% FBS. NSC were isolated and cultured as previously described (Di Stefano et al., 2009). All media were supplemented with L-glutamine and penicillin/streptomycin (GIBCO).
Extracted molecule
total RNA
Extraction protocol
RNA was isolated with the miRNeasy mini kit (Qiagen), eluted with RNase-free water and quantified by Nanodrop. cDNA was produced with the High Capacity RNA-to-cDNA kit (Applied Biosystem). RNA samples with a RIN greater than 9 were analyzed by expression arrays.
Label
Cy3
Label protocol
500ng of total RNA per sample were labeled using Agilent's QuickAmp labeling kit and hybridized to Agilent 8X60K expression arrays.
Hybridization protocol
not provided
Scan protocol
not provided
Data processing
Raw array data was processed using limma (Ritchie et al., 2015). We perform “normexp” background correction with an offset of 16. We normalize between arrays using quantile normalization. To identify differently expressed genes we create a contrast matrix in which all pairwise comparisons are made. For each probe an empirical Bayes moderated t-statistic test is performed. An FDR correction is applied to the nominal p-values.
