NextSeq 500 paired end sequencing; RNA-seq of whole animal tissue and AWA olfactory neurons isolated by FACS from control and post-dauer L4 larval stage C. elegans
RNA-seq of whole animal tissue and AWA olfactory neurons isolated by FACS from control and post-dauer L4 larval stage C. elegans
Animals were collected and cells dissociated essentially as described previously {Taylor, 2021}. In brief, animals were washed off plates with M9 buffer and incubated in lysis buffer (200 mM Dithiothreitol (DTT), 0.25% Sodium dodecyl sulfate (SDS), 20 mM HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 3% sucrose) for 5 mins. Lysed animals were washed 5X with egg buffer (118 mM NaCl, 48 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM HEPES, pH 7.3) following which the worm pellet was resuspended in 750 μl of 15 mg/ml Pronase (Sigma P8811) in egg buffer. To promote dissociation, the mixture was pipetted frequently for 20 mins and progress of dissociation was monitored under a light microscope. Pronase digestion was terminated by adding L-15-10 media (L-15 media + 10% volume FBS, HI) at 4℃. Dissociated worms were centrifuged at 4℃ and the worm pellet was resuspended in egg buffer and recentrifuged at 4℃. The supernatant was filtered through a 35 µm filter in preparation for FACS sorting. A 25 μl aliquot of the dissociated worm pellet was pipetted directly into Trizol for analysis as the whole worm sample. Prior to performing FACS, 0.5 μl of 1 mg/ml DAPI was added to 1 ml of the cell suspension to exclude dead cells. AWA neurons were captured by isolating the high GFP+ and low DAPI compartment and cells were sorted directly into Trizol. To obtain control animals, adult hermaphrodites expressing the stably integrated transgene gpa-4Δ6p::myrGFP (PY10421) were bleached, and eggs allowed to hatch in M9 with 0.1% Triton-100. 50,000-100,000 growth-synchronized L1 larvae were plated onto 15 cm 8P growth plates seeded with 1 ml of an overnight culture of E. coli NA22 grown in 2XYT media. L1 larvae were allowed to grow for 40-48 hrs at 20℃ to obtain populations of L4 larvae. To obtain post-dauer animals, SDS-selected dauer larvae were plated onto 15 cm 8P plates seeded with 1 ml of an overnight culture of E. coli NA22 grown in 2XYT media, and allowed to recover to the L4 stage for 20-24 hrs at 20℃. RNA was extracted from collection tubes using standard chloroform and isopropanol precipitation. The RNA pellet was resuspended in RNase-free water and any DNA digested using the RNase-Free DNase kit (Qiagen). RNA was eluted using the RNeasy MinElute Cleanup Kit (Qiagen). mRNA was isolated and libraries amplified using Smart-Seq v4 and library quality checked with Bioanalyzer using Agilent High Sensitivity Gel. Libraries were sequenced on Illumina NextSeq500. Library preparation and sequencing was performed at the MIT BioMicro Center (https://biology.mit.edu/tile/biomicro-center/).