Temperature analysis was done in three different ways: (1) standard TRACE analysis of the loop pixels with no background subtraction; (2) constant background subtraction for each TRACE image; (3) pixel pair background subtraction.
Intensities were quantified using QuantArray software utilizing automatic background subtraction for each spot.
After the background subtraction for each spot, the data were normalized to median, that is, the ratio of the median value of all spots in the Cy5 channel (IP DNA) and the ratio of the median value of the control channel (Cy3 Input).
This first set of images was used for background subtraction for each channel except for the DAPI channel.
Measurements were done in the entire growth cone area selected using the tyrosinated α-tubulin channel, after background subtraction for each channel.
In order to ensure statistical reliability for the evaluation of expression levels, three different concentrations of each gene probe were spotted in duplicate onto filters, background subtraction for each gene grid was performed, P-values were calculated taking into account all spots and selected experiments were repeated several times (Supplementary Fig. S1).
The sum of the myosin band pixel intensity (intensity x mm) in each lane was normalized to the corresponding actin intensity (adjusted after local background subtractions for each band) in the same lane.
Blanton, M. R., Kazin, E., Muna, D., Weaver, B. A. & Price-Whelan, A. Improved background subtraction for the Sloan Digital Sky Survey images.
This procedure allows the user to directly analyze the bead fluorescence to determine the SNP genotype, and it eliminates the need for background subtraction for signal determination.
Hopkins JB, Katz AM, Meisburger SP, Warkentin MA, Thorne RE, Pollack L, "A microfabricated fixed path length silicon sample holder improves background subtraction for cryoSAXS," Applied Crystallography Online 48(1), 227-237 (2014).
Hopkins JB, Katz AM, Meisburger SP, Warkentin MA, Thorne RE, Pollack L, "A microfabricated fixed path length silicon sample holder improves background subtraction for cryoSAXS," Applied Crystallography Online 48(1), 227-237 (2015).
