scRNAseq timecourse from wildtype C. elegans larvae sampled from 18 to 24 hours at 25oC, with FACS sorting cells using ubiquitous markers.
We collected a timecourse scRNA-seq dataset from wildtype C. elegans larvae, using ubiquitous flurophores to FACS sort cells in order to collect as many tissues as possible. The data deposited were collected from one single experiment with pools A,B,C representing different 10X runs for a given sample. The overall goal of the experiment was to identify the tissues in which oscillating gene expression occurs, and to understand tissue-specific differences in oscillatory output. We were able to identify 18 different tissues, 7 of which show clear oscillatory expression patterns. Overall design: HW3125 worms, expressing transgenes for ubiquitous GFP and mCherry expression, were synchrnoized as hatched L1 larvae. 200,000 worms per plate were seeded onto 16 peptone-rich XL plates coated with 10x concentrated OP50 and grown at 25oC. 600,000 worms (3 XL plates) were collected at each timepoint, 18h, 20h, 22h and 24h after seeding. At each timepoint, worms were immediately processed to isolate cells, FACS sort, and perform 10x scRNA-seq protocol. To dissociate single cells, we followed the protocol of Cao et al., 2017, Science) with minor adjustments. To remove animal debry, cells were then sorted on a BD FACSAria using the GFP and mCherry markers. Following sorting, cells were diluted and libraries prepared using the 10x Genomics protocol with the Chromium Controller (10x 3'RNAv3). Sequencing was then performed using NextSeq HIGH-OUT 75 cycle paired-end sequencing.