Cells were trypsinised, washed twice in PBS and resuspended at 2 x 106 cells/ml in FACS buffer (0.1% sodium azide, Sigma; 0.2% bovine serum albumin, Sigma; in PBS). Cells were labelled with antibodies at 4˚C for 1 hour at (unless stated otherwise) the following concentrations: mouse IgM anti-SSEA-1 directly conjugated to phycoeryithrin (PE; Santa Cruz) 2 mg/ml; mouse IgM conjugated PE isotype control (Santa Cruz) 2 mg/ml; 9A7 (rat IgG2A anti-m5T4) 20 μg/ml; rat IgG2A isotype control (eBioscience). 9A7 labelled cells were washed twice in 100μl FACS buffer, resuspended in the original volume of FACS buffer and incubated for 1 hour at 4˚C with rabbit anti-rat immunolglobulins directly conjugated to fluorescein isothiocyanate (FITC, 100 μg/ml, Dako Cytomation). Cells were washed twice and fixed as for SSEA-1 labelled cells. After fixing cells were analysed using FACScan flow cytometer running CellQuest software. A total of 20,000 events were collected and WinMDI 2.8 software (The Scripps Research Institute) was used to produce histograms of results. Live cells (prior to fixation) were gated using forward and side scatter, and this population used for analysis. Typically the gate was set so that it contained the upper 2.5% of the cells incubated with isotype control antibody. This was not practical in all cases due to long “tails” of the negative control histogram. In these instances the gated region was set based on the shape of the histogram. Fluorescence activated cell sorting (FACS): Cells were labelled with 9A7 as described for flow cytometry but the labelling incubation time was reduced to 45 minutes. Cells were not fixed but instead were filtered through a 30μm pre-separation filter (Miltenyi). Cells were sorted (into FACS buffer at 4˚C) using a FACSVantage SE cell sorter, having gated for live cells based upon forward and side scatter, and gating for cells with the same fluorescence intensity as the isotype control incubated cells (5T4 negative cells) and cells with higher fluorescence intensity than isotype control incubated cells (5T4 positive cells).
Growth protocol
Cells were grown in Falcon tissue culture flasks (Becton Dickinson) in a humidified atmosphere of 5% CO2 at 37°C. Tissue culture flasks were prepared for culture of ES cells by coating with 0.1% gelatin (Sigma) overnight at 4ºC. E14TG2a embryonic stem cells were grown in Knockout Dulbecco's modified Eagle's medium (DMEM; Invitrogen) supplemented with 10% Hyclone foetal calf serum (DMEM-FCS; Perbio) or 10% serum replacement (DMEM-SR; Invitrogen), 2mM L-glutamine, 1% non-essential amino acids (eBioscience), nucleosides [6 ml of the following solution/500 ml DMEM: adenosine (80 mg), guanosine (85 mg), cytidine (73 mg), uridine (73 mg) and thymidine (24 mg) dissolved in 100 ml double distilled water; Sigma], 2-mercaptoethanol (50 µM; Invitrogen) and leukaemia inhibitory factor (LIF; 1000 units/ml of ESGRO; Chemicon Int.). Media was changed daily. Cells were passaged every other day: growth medium was aspirated and cells washed in PBS. Cells were incubated with 1x trypsin-EDTA (Sigma) for 10s and incubated at 37ºC for 1 minute. Cells were resuspended in growth media and passaged 1:6. For differentiation of ES cells, cells were trypsinised and washed as described above. Cells were resuspended in media which was not supplemented with LIF and plated at the 4 x104 cells/cm2 . Media was changed daily. Cells were not passaged during differentiation.
Extracted molecule
total RNA
Extraction protocol
Prior to RNA extraction and subsequent processing, all apparatus and working surfaces were cleaned using RNaseZap (Ambion), to reduce the possibility of RNA degradation. After FACS sorting, cells were centrifuged (5 minutes, 350g) and resuspended in trizol (Invitrogen). RNA was extracted according to the manufacturer’s instructions. Briefly, cells suspended in trizol were incubated at room temperature for 5 minutes in RNA free 1.5 ml eppendorfs (Starlab). 0.2 ml chloroform (VWR) was added per 1ml trizol, shaken and incubated at room temperature for 2 minutes. Samples were centrifuged again (12000g, 15 minutes, 4˚C) and the upper aqueous phase transferred to a clean eppendorf and 0.5 ml propan-2-ol (VWR) per 1 ml trizol added. This was incubated at room temperature for 10 minutes, then centrifuged (12000g, 10 minutes, 4˚C). The supernatant was discarded and the RNA pellet resuspended in 75% ethanol (75% absolute ethanol, VWR, in RNase free water, Ambion). This was stored at -80˚C until ready for further analysis. When ready for further analysis the sample was removed from the freezer, centrifuged (7500g, 5 minutes, 4˚C) and supernatant removed. The pellet was allowed to air dry and resuspended in RNase free water. RNA was then cleaned using the RNeasy mini kit (Qiagen) according to the manufacturer’s instructions. 50 μl RNase free water (Qiagen) was used in the final elution from the column. RNA concentration was determined using a Nanodrop spectrophotometer with supplied Nanodrop 2.4.7c software (National Instruments). A260/280 ratios were calculated, and RNA samples with concentrations (determined using the Beer-Lambert law, with 40ng-cm/ml at λ=260nm used as the molar extinction coefficient of RNA) greater than 300ng/ml, and A260/280 ratios between 1.80 and 2.10 were deemed suitable for further analysis.
Label
biotin
Label protocol
RNA that passed the above criteria was prepared for microarray analysis, hybridised to the arrays and scannced by the Molecular Biology Core Facility, PICR. Before proceeding with labelling for microarray analysis, RNA quality and concentration was determined using an Agilent Bioanylyser (Agilent Technologies). In all cases satisfactory RNA integrity was observed. Biotinylated target RNA, for hybridisation to microarrays was prepared with minor modifications to the manufacturer’s instructions (Affymetrix). Spike controls were added to 10µg fragmented cRNA before overnight hybridisation to arrays. Full details of the protocol can be found in: http://bioinformatics.picr.man.ac.uk/mbcf/downloads/GeneChip_Target_Prep_Protocol-CR-UK_v3.pdf
