trr_WT_rep1
Drosophila melanogaster
source_name
Drosophila heads
tissue
head
developmental stage
adult (0-5 days old)
genotype
Elav-Gal4 / + ; dicer2 / + ; P{y[+t7.7]=CaryP}attP2 / +
extraction protocol
Total RNA was extracted from 50 µl aliquots of frozen fly heads using the RNAeasy lipid tissue mini kit (Qiagen). For trr knockdown, two biological replicates were used, for G9a mutants, three biological replicates were used for each condition. mRNA was purified using the Oligotex kit (Qiagen) and cDNA was synthesized using the SuperScript III First Strand synthesis kit (Thermo Fisher) using random hexamers as primers. Second strand cDNA was synthesized using E. Coli polymerase and T4 ligase (New England Biolabs Inc. (NEB)). Remaining RNA was removed using 2 units RNaseH (NEB) before the cDNA was purified using the MinElute PCR purification kit (Qiagen). DNA end repair was performed followed by ligation of Illumina sequencing adaptors and size selection for 300 bp by 2% E-gel (Invitrogen). Fragments were linearly amplified (15 PCR cycles), as validated by quantitative real-time PCR (qPCR), and sample quality was assessed using the Agilent Bioanalyser 2100.