RNA-seq of regenerated Earthworm-anterior-Blastema-tissue-4th-day-10th-Segment-cut
10 ug of total RNA was used to prepare the RNA seq library using the TruSeq RNA Sample Prep Kit v2 (Illumina, San Diego, CA). The libraries were prepared as per the kit protocol. Briefly, polyA containing mRNA molecules were purified using the polyT oligo attached magnetic beads. Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were used to synthesize first strand cDNA using reverse transcriptase and random primers followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments were subjected to an end repair process, the addition of a single A base, and then ligation of the adapters. The products were purified and enriched with PCR to create the final cDNA library. After the libraries were constructed, paired end run was performed on Illuminas HiSeq 2500 platform to generate 20-25 million paired end Reads Per sample.