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Analysis time between each sample was reduced to less than 20 s, all target molecules being analyzed in a single run with the use of laser diode thermal desorption atmospheric pressure chemical ionization (LDTD/APCI) coupled with high resolution accurate mass (HRMS) orbitrap mass spectrometry.
The protein concentration between each sample was consistent, which is confirmed by comparable signal intensities in the Coomassie-stained gels.
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To correct gene models and to compare the expression between samples, each sample was first mapped against its reference genome using TopHat [ 89] (version v2.0.6, parameter -g ).
To allow for multiple comparisons between gels, each sample was compared to its respective 0 h that was run on the same gel.
Tumor and matched normal samples were always analyzed in the same run to exclude between-run variations and each sample was studied in duplicate.
The continuous band occurring in the range of frequencies between 3000 3700 cm−1 for each sample was fitted to theoretical one.
To calculate ratios between different elements, each sample was measured six times.
The average methylation between G and A-alleles in each sample was between 35 and 55% consistent with the total methylation levels obtained with our standard pyrosequencing assay.
We estimate that the sorting time for each sample was between 1 and 5 min, depending on the abundance of each subpopulation being isolated at each time point.
The distance between the laser source and each sample was fixed to be 2 cm.
The temperature difference ΔT between the two ends of each sample was controlled at 4-6°C 4-6°Crying the flowing rate of Ar gas.
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