title: GSM1548146: 114; Penicillium oxalicum; RNA-Seq
library_construction_protocol: Total RNA was extracted, and oligo (dT) beads were used to purify mRNA and guide double-stranded cDNA synthesis. The enzyme NlaIII was used to cut the cDNA at CATG sites. After Illumina adapter 1 was linked to the CATG site, MmeI was used to cut at 17-bp downstream of the CATG site. Then Illumina adapter 2 was linked at 3' end and the fragments were PCR amplified. Finally, the 85-bp strips were gel purified and digested for Solexa sequencing. Each tunnel generated millions of raw reads with sequencing length of 35bp. 3' adaptor sequences were first removed from the sequencing reads to produce 21-nt long tags. Then low quality tags, one copy tags and tags which are not 21-nt long were removed to generate clean tags. Reference 21-nt tag databases were created from predicted P. decumbens transcripts plus downstream 300-nt sequences of two strains. All clean tags were mapped to corresponding reference tag databases and no mismatch was allowed. The copy number of unambiguous tags (tags mapped to one single gene) for each gene was normalized to TPM (number of transcripts per million clean tags) for differential expression analysis.