title: GSM2882633: WT_Amputated_36hr-3; Schmidtea mediterranea; RNA-Seq
library_construction_protocol: For untreated and 1-day irradiated animals, total RNA was purified using Trizol as described (Zeng et al., 2013). For sorted X1, X2 and Xins populations based on Hoechst staining, 1x105 cells were sorted into 800 μl Trizol LS, and RNA was extracted according to the manufacturer's manual. RNA from whole worms was treated with RNase-free DNase on QIAGEN RNAeasy columns and was eluted in nuclease-free water (Ambion). For each replicate, 500ng-1μg RNA from five worms or 100 ng RNA from 1x105 sorted cells were used to generate RNA-Seq libraries using the Illumina TruSeq Stranded kit. For RNA extraction from fixed cells, total RNA was isolated from the pellet using the RecoverAll Total Nucleic Acid Isolation kit (Ambion), starting at the protease digestion stage of manufacturer-recommended protocol. The following modification to the isolation procedure was made: instead of incubating cells in digestion buffer for 15 minutes at 50°C and 15 minutes at 80°C, we carried out the incubation for 1 hours at 50°C. Cell lysates were frozen at −80°C overnight before continuing the RNA isolation by the manufacturer's instructions. For each replicate, 500ng-1μg RNA per whole worm sample or 100ng of sorted cell RNA was used for generation of RNAseq libraries using the Illumina TruSeq kit. cDNA quality was determined by Agilent high sensitivity DNA kit on Agilent 2100 BioAnalyzer (Agilent Technologies).
attributes.tag: GEO Accession
attributes.value: GSM2882633