title: GSM8397463: LHLK, 24-hr fed or starved, batch 1; Drosophila melanogaster; RNA-Seq
library_construction_protocol: In brief, after tethering cold-anesthetized fly on a custom fly stage and immersing the fly head in isotonic PBS, microdissection was conducted to remove the posterior dorsal lateral head cuticle to allow access to the LHLK neurons. A custom rig equipped with micromanipulator and infra-red and fluorescent DIC and glass electrodes were used to harvest the individual LHLK neurons from both sides of the animal's brain. After harvesting each LHLK neuron, the tip of electrode was broken into a microcentrifuge tube prefilled with PBS with RNase inhibitor. The tube was briefly mixed, flash frozen in liquid nitrogen, and stored at –80 ℃ before library construction. Sampling between starvation and control groups was performed in an alternating manner. CEL-Seq2 protocol. In brief, 18 to 28 harvested LHLK neurons were processed in parallel for individual library constructions. Single-cell RT was conducted with primers that contain sequences of Illumina RNA 5' adaptor (RA5), hexamer UMI, single-cell barcode, and poly-T ending with V (not T), followed by second-strand synthesis. Then, all the samples were pooled together for in-vitro transcription-based amplification. A second round of RT was conducted with a primer that contain 3'-end common sequence of Illumina RNA PCR Index Primer sets and random hexamer, followed by 11-cycles of PCR. After 2 rounds of bead cleanup, the libraries were quantified by Qubit, size-profiled by Bioanalyzer, and ready for sequencing