GSM3559473: m2-73_w_2; Drosophila melanogaster; RNA-Seq
from each genotype, ~50 wing discs were dissected into Trizol reagent (25 discs into 250μL) and frozen at -80°C. Following thawing and centrifugation (12000g 15 min 4°C), the aqueous phase was precipitated with an equal volume of isopropanol (10 min room temperature). The pellet was recovered by centrifugation (12000g 15 min 4°C), washed with RNAse-free 75% ethanol twice, and air dried (5-10 min room temperature) after removing the remaining ethanol with a filtered RNAse-free pipette tip. Samples were resuspended in RNAse-free water, analyzed by nanodrop spectroscopy and Bioanalyzer and satisfactory samples stored at -80°C library was constructed by Beijing Genomics Institute. After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) are used to isolate mRNA(for eukaryotes) or by removing rRNAs from the total RNA (for prokaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short fragments are connected with adapters. After agarose gel electrophoresis, the suitable fragments are selected for the PCR amplification as templates. During the QC steps, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library.
GEO Accession
GSM3559473