Transcriptome responses of earthworms to mineral weathering
The total RNA samples were examined for purity and integrity with 1% agarose gel electrophoresis, spectrophotometry (NanoDrop ND-1000), and Bioanalyzer electrophoresis (Bioanalyzer 2100, Agilent Technologies).  For each treatment, the RNA samples from the three biological replicates were then pooled together in equal amounts to generate one mixed sample. These three mixed RNA samples were used in sequencing library construction with Illumina TruSeq™ RNA Sample Preparation Kits (Illumia, San Diego, USA). Library construction was done following the instructions of the manual through the following steps: rRNA removal, fragmentation, first strand cDNA synthesis, second strand cDNA synthesis, terminal repair, 3’-end A-tailing, adapter ligation, enrichment.  The libraries were measured for concentration with a Qubit® 2.0 Fluorometer (Invitrogen), and examined for sizes with an Agilent 2100 Bioanalyzer. Cluster generation and the first sequencing primer hybridization were done using the cBot (Auxiliary of Illumina HiSeq 2500 sequencer). The flow cell containing the cluster was then set in the Illumina HiSeq 2500 to conduct paired end 2 x 100 nt multiplex sequencing.