title: RNA-seq of all C. elegans cells from etr-1 mutants
design: Larval disruption for cell sorting. For body wall muscle cell sorting, the myo-3::gfp transgene ccIs4521 was used, in wild-type and etr-1(lq61) backgrounds. C. elegans strains were grown on twenty x 150 mm 8P nutrient agar plates (for 1 liter: 20 g bactopeptone, 3 g NaCl, 25 g agar) with E. coli strain NA22, which produces a thick bacterial lawn. Early L1 animals were synchronized by first collecting embryos using bleach (hypochlorite) treatment of gravid adult hermaphrodites, and allowing the embryos to hatch overnight into sterile M9 medium at 20C. This produced approximately 3 million starved, synchronized early L1 larvae. The Q neuroblasts of starved L1s were variably arrested in their early development, from initial migration to the first division after migration (data not shown). Preparations of dissociated larval cells were generated as previously described using SDS-DTT and Pronase treatment coupled with mechanical disruption using a pipet (Spencer et al., 2014).FACS analysis. Fluorescence activated cell sorting of myo-3::GFP-expressing body wall muscle cells was performed as previously described using a BD FACSAria with a 70 micron diameter nozzle (Spencer et al., 2014). DAPI was added to mark dead or damaged cells which were excluded from each sample of viable myo-3::GFP labeled muscle cells. Profiles of GFP strains were compared to a non-transgene-bearing N2 standard to exclude auto-fluorescent cells. Cells were sorted into Trizol LS for RNA extraction. 30,000-50,000 FACS-sorted cells from each experiment, yielded 5-100 ng total RNA which was used in RNA seq library preparation. Three independent replicate samples were obtained for both wild-type and etr-1(lq61) mutant animals.mRNA Library Prep and Sequencing MethodsRNA was isolated from twelve RNA samples representing three biological replicates of N2 and etr-1(lq61) sorted muscles and three biological replicates of N2 and etr-1(lq61) sorted whole animal cells collected from the same cell population after muscle cells had been sorted. Quality of total RNA samples was ensured using an Agilent TapeStation, and quantity was determined using Qubit fluorimetry. Sequencing libraries were constructed with 5-100ng of total RNA using the NEBNext Ultra II Directional RNA Library Kit for Illumina. The sequencing library construction process aimed for 300-bp inserts and included mRNA purification with poly-A beads, fragmentation, strand specific cDNA synthesis, end repair, 3 end adenylation, adapter ligation, and PCR amplification. The constructed sequencing libraries were validated and quantified with Qubit and TapeStation assays. Each library indexed and sequenced in multiplex on the Illumina NextSeq550 system, generating paired-end, 150-base sequence reads from the libraries. Between 33 million and 39 million reads were generated for each of the twelve samples. Base calling was carried out by the Nextseq550 instrument Real Time Analysis (RTA) software. The base call files (bcl files) were demultiplexed and converted to compressed FASTQ files by bcl2fastq2.