title: GSM9305641: Command interneurons, L1 stage hermaphrodite Caenorhabditis elegans, 5929; Caenorhabditis elegans; RNA-Seq
library_construction_protocol: Fluorescence Activated Cell Sorting (FACS) was performed on a BD FACSAria™III Cell Sorter equipped with 70-micron diameter nozzles. DAPI was added to the sample (final concentration of 1 μg/mL) to label dead and dying cells. We used a variety of fluorescent reporter strains to label subsets of neuronal cells. N2 worms lacking fluorescence and single-color controls (for intersectional labeling strategies and dual-color sorts) were used to set gates to exclude auto-fluorescent cells and to compensate for spillover between fluorescent channels. In three experiments, single-cell suspensions from separate strains were combined prior to FACS: (1) NC3583, NC2957; (2) OH16003, EG1285, ZM5488 and uIs152; (3) CZ631, NC3523, PS3504. Cells were sorted under the “4-way purity” mask. Cells expressing the desired fluorescent proteins were collected into 400 μL of chilled (4o C) L-15 media containing 33% FBS (L-15-33) and stored on ice. We concentrated the collected cells by centrifuging at 500 rcf for 5 minutes at 4o C. We removed all but ~30-50 μL of the supernatant, resuspended the cells and counted fluorescent cells on a hemocytometer. Cell suspensions loaded into the 10X Chromium Controller ranged in concentration from 350-1000 cells/uL. Libraries were prepared using P/N 1000121 following the manufacturer's protocol. The libraries were sequenced using the Illumina NovaSeq 6000 with 150 bp paired end reads.