Cells were washed with PBS and then harvested by trypsinization for RNA extraction
Growth protocol
Isolating and expanding naïve human pluripotent cells: The following serum free defined conditions, termed WIS-NHSM (Weizmann Institute of Science Naïve Human Stem cell Medium) were used to isolate, generate, derive and stabilize naïve human pluripotent stem cells (iPSCs and ESCs) with the unique biological properties described in this study. WIS-NHSM media was generated by including: 475 ml Knockout DMEM (Invitrogen 10829), 5gr AlbuMAX (Invitrogen 11020-021), 5 ml N2 supplement (Invitrogen 17502048), 10 µg of recombinant human LIF (Peprotech), 8ng/ml recombinant bFGF (Peprotech) and 1ng/ml recombinant TGFβ1 (Peprotech), 1 mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1mM β-mercaptoethanol (Sigma), Penicillin-Streptomycin (Invitrogen) and small molecule inhibitors: PD0325901 (1 µM, ERK1/2i, AXON MEDCHEM); CHIR99021 (3µM, GSK3bi, AXON MEDCHEM); SB203580 (5µM, p38i, TOCRIS); SP600125 (10µM, JNKi, TOCRIS). Throughout the study Naïve hESCs/hiPSCs were grown on 0.2% gelatin + 1ng/ml vitronectin coated plates for at least 1 hour in 37oC. Cells were passage by single cell trypsinization (0.25% or 0.05% Trysin+EDTA) every 3-4 days. Although not essential, enhanced single cell cloning efficiency can be obtained with WIS-NHSM supplementation with ROCK pathway inhibitor Y-27632 (5 µM, ROCKi Axon Medchem) for 24 hours before and after cell passaging. Culture of conventional/primed human ESCs and iPSCs:The following already established conventional human ESCs and iPSC lines were used (indicated passage number of the cell line taken for conversion into naïve pluripotency is indicated in parentheses): Human induced pluripotent stem cells C1 (P21) and C2 (P9) hiPSC lines 15 and the human embryonic stem cell (hESC) lines BGO1 (P35) (National Institutes of Health ID code BG01; BresaGen], H1 (P40), H9 (P37), WIBR1 (P13), WIBR3 (P11) hESCs were maintained in 20% pO2 conditions (unless indicated otherwise) on irradiated mouse embryonic fibroblast (MEF) feeder layers or Gelatin/vitronectin coated plates, in hESC medium: (425ml Knockout-DMEM – Invitrogen 10829) supplemented with 15% Knockout Serum Replacement (Invitrogen 10828-028), 1 mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma), and 8 ng/mL bFGF (Peprotech) and 1 ng/ml recombinant human TGF β 1 (Peprotech). Cultures were passaged every 5–7 days either manually or, or by trypsinization (24 hour pre and 24hour after addition of ROCK inhibitor at 10nM concentration).
Extracted molecule
total RNA
Extraction protocol
mRNA was extracted byTRIZOL reagent (invitogene) according to manufacturer's instructions
Label
Biotin
Label protocol
250 ng total RNA was used to prepare biotinylated aRNA (cRNA) according to the manufacturer’s protocol (Affymetrix 3’ IVT Express Kit). Briefly, total RNA undergoes T7 oligo(dT)-primed reverse transcription to synthesize first-strand cDNA containing a T7 promoter sequence. This cDNA is then converted into a double-stranded DNA template for transcription using DNA Polymerase and RNase H to simultaneously degrade the RNA and synthesize second strand cDNA. In vitro transcription synthesizes aRNA and incorporates a biotin-conjugated nucleotide. The aRNA is then purified to remove unincorporated NTPs, salts, enzymes, and inorganic phosphate. Fragmentation of the biotin-labeled aRNA prepares the sample for hybridization onto GeneChip 3’ expression arrays.
Hybridization protocol
Samples were prepared for hybridization using 12.5 µg of biotinylated aRNA in a 1X hybridization cocktail according the Affymetrix hybridization manual. Additional hybridization cocktail components were provided in the Affymetrix GeneChip Hybridization, Wash and Stain Kit. GeneChip arrays were hybridized in a GeneChip Hybridization Oven at 45°C for 16 hours at 60 RPM. Washing was done using a GeneChip Fluidics Station 450 according to the manufacturer’s instructions, using the buffers provided in the Affymetrix GeneChip Hybridization, Wash and Stain Kit.
Scan protocol
Arrays were scanned on a GeneChip Scanner 3000 and images were extracted with Affymetrix GeneChip Command Console (AGCC), and analyzed using GeneChip Expression Console.
Description
Gene expression data of the indicated cell line
Data processing
The data were analyzed with Matlab software and its Bioinformatics toolbox, using RMA method.
