Illumina MiSeq paired end sequencing; Genome-wide analysis of Groucho in Drosophila Kc167 and S2R+ cell lines
Genome-wide analysis of Groucho in Drosophila Kc167 and S2R+ cell lines
Kc167 and S2R+ cells (obtained from the Drosophila Genomics Resource Centre [DGRC]) were grown in Schneiders Insect Cell Medium (GIBCO) supplemented with 10% FBS (Sigma) and 1% penicillin/streptomycin (Thermoscientific). Cell lines were kept at 25C. For Groucho oligomerization experiments cells were cotransfected with dsRNA against the 3UTR of Gro as well as plasmids containing either Gro-GFP or GroL38D;L87D-GFP. Transfections were performed using Effectene according to manufacturers instructions (Qiagen). 300-600ng DNA and 1 microgram of dsRNA were cotransfected per 1-3 x 10^6 cells. Successfully transfected cells were processed six days post transfection. For RNA-seq experiments total RNA was obtained from approximately 5 x 10^6 cells per sample using the Qiagen RNeasy mini kit. mRNA was then extracted using the Dynabeads mRNA Purification Kit (Life Technologies). 50-250 ng of mRNA was used to generate an mRNA library following manufacturers instructions (NEBnext mRNA Library Prep Master Mix _ E6110S). cDNA fragments of 250-350 bp were selected by agarose gel extraction and amplified with 12 cycles of PCR. Resulting libraries were assessed for concentration and size using the Qubit fluorometer and the Agilent2100 Bioanalyzer.