title: GSM9060681: adult brain, pcm-RNAi CT12 repeat-1; Drosophila melanogaster; RNA-Seq
library_construction_protocol: Flies of genotype ClkGAL4>UAS-tetR-GFP-NLS was used for a bright GFP marker specific to clock neurons and DAPI was used as a viability marker. A small population of cells were heated at 60C for 5min and used as a dead cell (DAPI+) control. Live clock neurons are defined by GFP+ and DAPI- gating. For each sample, ~2,000 live clock were sorted with FACS sorter (Sony MA900) directly into cell lysis buffer (NEBNext Low-Input cDNA Synthesis Module) in 1.5mL DNA low-bind tube (Eppendorf). Final volume of the lysis buffer (with sorted cells) is ~16uL. Cells were lysed and half of the lysate was subsequently used for cDNA library preparation following manufacturer protocol. cDNA library was subject to quality control by Tapestation (HSD5000) and Qubit (High Sensitivity DNA) for mean size of ~1500bp. Next, cDNA library was tagmented to prepare Illumina sequencing library (Illumina Nextera XT, UDI indexing). Quality control was performed with Qubit (High Sensitivity DNA) and Tapestation (HSD1000) for mean size of ~300bp. Control/pcm-RNAi samples were loaded in equal molar with 3% spike-in PhiX DNA and sequenced on an Illumina NextSeq 1000 platform with paired-end 100bp cycle using P2 flow cell. Control/me31B-rABE samples were sequenced similarly on an Illumina NextSeq 1000 platform with paired-end 300bp cycle using P2 flow cell.