GSM4006768: lateral_int_rep1; Schmidtea mediterranea; RNA-Seq
Frozen, stained PEN slides were warmed to room temperature in 100% ethanol to maintain RNAse inactivation during thawing, then air dried for 2-3 minutes. Tissue was captured using a Leica LMD7 laser microdissection microscope at 10X magnification using the following parameters: Power-30; Aperture-20; Speed-5; Specimen Balance-1; Head Current-100%; Pulse Frequency-392 Hz. Regions were dissected into empty RNAse-free 0.5 ml microcentrifuge caps. After capture, 20 µl Buffer XB (Arcturus PicoPure RNA Isolation Kit) was added to captured tissue, then tubes were immediately frozen on dry ice and stored at -80°C prior to RNA extraction. Total RNA was extracted using the Arcturus PicoPure RNA Isolation Kit, quantified using a Denovix UV Spectrophotometer, and analyzed using Agilent HS RNA ScreenTape on an Agilent TapeStation 2200. Libraries were generated using the Lexogen Quantseq 3' mRNA Library Prep Kit according to the manufacturer's protocol, with 5 ng total RNA input for each sample. Briefly, first-strand cDNA was generated using 5'-tagged poly-T oligomer primers. Following RNase digestion, second strand cDNA was generated using 5'-tagged random primers. A subsequent PCR step with additional primers added the complete adapter sequence to the initial 5' tags, added unique indices for demultiplexing of samples, and amplified the library. Final libraries for each sample were assayed on the Agilent Tapestation for appropriate size and quantity.
GEO Accession
GSM4006768