title: Mus musculus musculus extra-orbital lacrimal gland partial (400-1300bp) transcriptome from 1 feral male
design: RNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufactures protocol with on-column DNase I treatment. cDNA was prepared using the SMARTer PCR cDNA Synthesis Kit (Clontech) and amplified with Advantage 2 PCR Kit (Clontech). Both procedures were handled according to protocol for Trimmer-2 Normalization Kit (Evrogen). The products of optimalized cDNA amplification were then loaded on AGE. For each sample, only the area of product in range from ~400bp to ~1300bp (well visible area full of bands) was excized from the gel and the DNA products were extracted using the Gel/PCR DNA Fragments Extraction Kit (Geneaid). Appropriate amount of size-selected products were then secondarily amplified according to the noted protocol from Evrogen. Products of secondary amplification were purified using MiniElute PCR Purification Kit (Qiagen). Rapid Library (RL) was prepared for each transcriptome (4 males and 4 females) according to Rapid Library Preparation Manual (my454.com). Equal amounts from each of 8 Rapid Libraries (107 molecules per µl dilution) were mixed and then used for emPCR. Further steps were following the provider´s instructions for sequencing with GS Junior (Roche; emPCR Amplification Method Manual Lib-L and Sequencing Method Manual, my454.com).