Quantity one 1 d

RNA was isolated from 0.5 g of each freeze-dried sample, according to the hexadecyltrimethylammonium bromide (CTAB) buffer lysis method, followed by chloroform/isoamyl alcohol extraction and overnight precipitation with lithium chloride (LiCl) proposed by van Dijk et al. (2009) [14 (link)], with some modifications, as follows. Lysis was performed with the extraction buffer pre-warmed to 60°C before use; the chloroform/isoamyl alcohol extraction was repeated three times before the LiCl precipitation; and the final precipitation with 96% ethanol was performed with the tubes kept on ice and then centrifuged at 4°C for 15 min at 14,000 g. Total RNA isolated was dissolved in 100 μL of 10 mM Tris (pH 7,0) and warmed to 65°C for 10 min. Total RNA was stored at -80°C until use.
RNA purity and concentration were assessed by absorbance measurements using a Nanodrop 1000 instrument (Thermo Fisher Scientific, NanoDrop Technologies Wilmington, DE, USA). For integrity evaluation, 1 μg of RNA was migrated by electrophoresis (10 min at 80 V and 50 min at 100 V) in denaturing agarose gel (1% agarose, 5% formamide, 1X TBE) stained with ethidium bromide. Gels were visualized in Gel Doc XR+ Systems (Bio-Rad Laboratories, Life Technologies Corporation, Carlsbad, CA, USA) and analyzed using Quantity One 1-D (Bio-Rad Laboratories).

ImageJ 1.8 software (U.S. National Institutes of Health, Bethesda, MD, USA) [66 ,67 (link)] was used to compare the intensities of the pDNA and RNA bands on the agarose gels, where the top and bottom phases of the second ATPS (20% (w/w) PEG 600 and 15% (w/w) ammonium sulphate were analysed. The images of the gels collected from the gel documentation software Quantity One 1-D (Bio-Rad) were used for this analysis. The recommendations in the ImageJ User Guide for densitometry were followed, with some modifications, as described next. First, the images were adjusted to the 32-bit mode and the look-up table (LUT) was inverted. Then, the lanes of the gels containing the corresponding pDNA and RNA bands were selected using the Rectangular Selection tool. The profile plot representing the relative densities of the bands contained in the selected area was obtained using the Plot Lanes function of the Analyze Gels tool. The area of each peak, corresponding to each band, was measured with the help of the Wand tool. The Label Peaks function (Analyze Gels tool) was used to label each peak with its size, expressed as a percentage of the total size of all of the analysed peaks.

All products of the time‐course reactions were stopped with 4xLaemmli sample loading buffer (Bio-Rad), resolved by 12% SDS–PAGE (in reducing or non-reducing conditions as indicated in the figures legends) and visualized by Alexa488 fluorescence emission in a Molecular Imager Versadoc MP4000 System (Bio‐Rad). Band densitometry was calculated by Quantity One 1-D (Bio-Rad).

Images of the 2DE Western blots were saved as TIFF files for analysis using Quantity One 1-D and spot identification with PDQuest analysis software (Bio-Rad). Protein density was quantified by dividing each membrane into a 7 x 23 grid of 161 equal sized rectangles that were placed on the identical positions on each individual image of the 2DE Western blots based on pI and MW. The average density of each rectangle was used to determine whether Sp185/333 proteins with varying pI identified in multiple sea urchins differed in prevalence within a given MW region on a blot. Duplicate samples from the same sea urchin were run and analyzed in parallel to ensure that the observed shifts were not artifacts of protein spreading in 2DE that resulted from sample processing (S1 Protocol; S3 Fig).

The DGGE fingerprints were analyzed using a Quantity One 1D software (BioRad). The total number of DGGE bands was used to represent OTUs richness (Duarte et al., 2009 (link)). Bacterial diversity was estimated based on densitometric measurements and Shannon diversity index (H′) (Duarte et al., 2009 (link); Ping et al., 2010 (link)), Equation (1):
P_i is a relative intensity of DNA band in the fingerprint, n_i is densitometrically measured intensity of individual DNA band, and N_i is the total amount of DNA in the fingerprint. The relative intensity of each band (P_i) was used to express the relative frequency of each phylotype (Moreirinha et al., 2011 (link)).