RNA was purified using the RNeasy mini kit (QIAGEN, Alameda, CA) following the “RNA Clean Up” protocol. After purification, the RNA concentration of each sample was measured with a Nanodrop® spectrophotometer (Thermo Scientific, Wilmington, DE) and total

RNA quality was checked by electrophoresis. Libraries prepared from bacteriome tissue SO (symbiont-full bacteriome) and AO (symbiont-free bacteriome) Libraries (see Table 1) were prepared using the Creator SMART cDNA Library Construction kit (Clontech/BD Biosciences, PaloAlto, CA), following the manufacturer’s instructions. cDNA was digested with Sfi1, purified (BD Chroma Spin – 400 column) and then ligated into a pDNRlib vector for E. coli transformation. SSH SSHA (symbiont-full/symbiont-free bacteriome), SSHB (symbiont-free/symbiont-full Milciclib bacteriome), SSH1 (Challenged/Non-RGFP966 Challenged with

S. typhimurium) and SSH2 (Non-Challenged/Challenged with S. typhimurium) www.selleckchem.com/products/ew-7197.html were performed by Evrogen (Moscow, Russia). In order to reduce the number of false-positive clones in the SSH-generated libraries, the SSH technology was combined with a mirror orientation selection procedure [38]. Purified cDNA were cloned into the pAL16 vector (Evrogen, Moscow, Russia) and used for E. coli transformation. Normalized library NOR was prepared by Evrogen (Moscow, Russia). Total RNA was used for ds cDNA synthesis using the SMART approach [39]. SMART prepared amplified cDNA was then normalized according to [40]. Normalization included cDNA denaturation and reassociation, using treatment with duplex specific nuclease (DSN), as described by [41]. Normalized cDNA was purified using a QIAquick PCR Purification Kit (QIAGEN, Alameda, CA), digested with restriction enzyme Sfi1, purified (BD Chroma Spin – 1000 column), and ligated into a pAL 17.3 vector (Evrogen, Moscow, Russia) for E. coli transformation. EST sequencing and data processing All clones from the libraries were sequenced

for using the Sanger method (Genoscope, Evry, France) and were deposited in the GenBank database. A general overview of the EST sequence data processing is given in Figure 1. Raw sequences and trace files were processed with Phred software [42, 43] in order to remove any low quality sequences (score < 20). Sequence trimming, which includes polyA tails/vector/adapter removal, was performed by cross_match. Chimerical sequences were computationally digested into independent ESTs. Figure 1 Sequence treatment (A) and functional annotation procedure (B). Clustering and assembly of the ESTs were performed with TGICL [44] to obtain unique transcripts (unigenes) composed of contiguous ESTs (contigs) and unique ESTs (singletons). For this purpose, a pairwise comparison was first performed using a modified version of megablast (minimum similarity 94%). Clustering was performed with tclust, that works via a transitive approach (minimum overlap: 60bp to 20bp maximum from the end of the sequence).