The expression levels of each contig were calculated and normalized to RPKM (Reads Per Kilobase of exon model per Million total reads in sample). In the RNA-seq-Cd.xls file, expression abundance (RPKM) of each 57162 contigs is present. CK1-CK3, three replicates of untreated sample; Cd1-Cd3, three replicates of treated sample.
cDNA library was constructed and sequenced with Illumina Solexa sequencing. After removing the low quality reads, the clean reads from Solexa sequencing in each sample were mapped to the assembled contigs and normalized to RPKM.
Total RNA was extracted and quality checked with Agilent 2100 Bioanalyzer (Agilent, USA). mRNA was purified with Micropoly(A)PuristTM mRNA purification kit (Ambion, USA) following the DNase I digestion (Ambion, USA). cDNA was synthesized with GsuI-oligo dT primer and Superscript II reverse transcriptase (Invitrogen).
S. alfredii Hance (HE) were obtained from an old Pb/Zn mine area in Zhejiang Province in China and cultured hydroponically in basal nutrient solution supplied with or without 100 uM CdCl2 for 8days.