GSM9489030: Shg_rep3; Drosophila melanogaster; RNA-Seq
Newly emerged flies expressing UAS-GFP and E-cadherin RNAi driven by c587ts in IGS cells were cultured for 1 week at 29°C. The ovaries were then dissected, placed in Grace's medium, washed twice with 1X DPBS, and centrifuged at 700 g for 1 minute. Next, the ovaries were incubated in prewarmed collagenase solution at 37°C in a water bath for 3 minutes with gentle shaking, after which the enzymatic reaction was stopped by adding cold 1X DPBS containing 2% FBS. The dissociated samples were washed with 1X DPBS and centrifuged at 700 g and 4°C for 5 minutes. The cell pellet was resuspended in 1X DPBS, filtered through a 70 µm cell strainer, pelleted again by centrifugation, and finally resuspended in 200 µl of 1X DPBS for immediate sorting of GFP-positive cells. 1,000 cells from each of three biological replicates for both the control and E-cadherin RNAi groups were collected. cDNA was synthesized using the SMART-seq v4 Ultra Low Input RNA kit, and libraries were prepared with the Nextera XT DNA Sample Preparation Kit and Index kits. Both cDNA samples and libraries were quality-checked on a Bioanalyzer 2100 before RNA sequencing. Libraries were sequenced as 75-bp paired-end reads on an Illumina NextSeq platform, generating over 20 million FASTQ reads per sample.