GSM8449895: C. elegans, C.chinensis extract, biol rep 2; Caenorhabditis elegans; RNA-Seq
On the 12th day of adulthood, the worms were collected and transferred to lysis tubes (around 3000-4000 worms per tube) with steel beads and stored at -80 °C. The RNA extraction proceeded according to the kit's manufacturer (innuSPEED Tissue RNA Kit by Analytik Jena Germany). The Turbo DNA-free kit from Thermo Fischer Scientific (Germany) was used at the end to degrade any possible DNA within the samples. The RNA content of each sample was measured with a Nanodrop and the condition of the RNA was checked with gel electrophoresis. The preparation of RNA library and transcriptome sequencing was conducted by Novogene Co., LTD (Beijing, China). Briefly, a total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using PE Cluster Kit cBot-HS (Illumina) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on an Illumina platform, and paired-end reads were generated.