Single-cell RNA sequencing of Caenorhabditis elegans L4-stage larvae

Methods

Synchronized L4-stage larvae of Caenorhabditis elegans (strain N2) were washed three times in M9
buffer and dissociated at 20 C using a combination of SDS-DTT treatment and pronase digestion.
Cell suspensions were passed through a 5 um filter and viability was confirmed by trypan blue
exclusion (>90% viable). Approximately 12,000 cells were loaded per lane with a target recovery of
7,000 cells.

Libraries were prepared using the Chromium Single Cell 3' v3 Reagent Kit according to the
manufacturer's instructions. Reverse transcription was carried out for 45 min at 53 C, followed by
12 cycles of cDNA amplification. Final libraries were quantified by Qubit and sized on an Agilent
Bioanalyzer, then sequenced on an Illumina NovaSeq 6000 with 28 cycles for read 1 and 91 cycles for
read 2.

Reads were aligned to the WBcel235 reference assembly. Cells with fewer than 200 detected genes or
more than 25% mitochondrial counts were excluded from downstream analysis.
