GSM5150363: AX7866 d4_1; Caenorhabditis elegans; RNA-Seq
Synchronised transgenic young adults in which the ADL neurons were specifically labelled using srh-220p:mKate2 were washed 5x in M9 Buffer to remove bacteria and then dissociated as described (Beets et al., 2020; Kaletsky et al., 2018). Briefly, animals were incubated for 6.5 min in Lysis Buffer (200mM DTT, 0.25% SDS, 20mM HEPES, 3% sucrose) washed 5x in M9 Buffer, resuspended in 500 μL of 20 mg/uL Pronase in water, and pipetted up and down for 12 min at room temperature. The reaction was stopped by adding 250 μl of 2% FBS in PBS. Cells were filtered through a 5 μm syringe filter to remove clumps. mKate positive cells were sorted using a Synergy High Speed Cell Sorter (Sony Biotechnology) with gates set using a negative control prepared in parallel from dissociated unlabelled N2 animals. Positive cells were collected into 10 μL of Triton X-100 0.2% (vol/vol) supplemented with 2U/mL RNase inhibitors. Between 700 and 3000 cells were collected for each biological replicate. Library preparation of sorted neurons was adapted from Picelli et al., . Briefly, fluorescently-labelled neurons collected by FACS were lysed in 10 μL of 0.2% Triton X-100 (vol/vol) and 2U/μL RNase inhibitors. The reverse transcription reaction volumes were adjusted to a 10 μL input and cDNA prepared using oligo dT primers and template-switching oligos (TSO) to enrich for polyadenylated transcripts and allow for pre-amplification of cDNA. cDNA was pre-amplified using custom PCR primers. 50 μL of PCR product from the pre-amplification step were purified using 50 μL of Ampure XP beads (Beckman Coulter), resuspended, and used at a concentration of 0.2 ng/uL as input for library preparation using the Nextera XT DNA Kit. Library preparation followed the manufacturer's instruction. The quality of RNAseq libraries was assessed on a Bioanalyzer (Agilent) using High Sensitivity Gels (Agilent). Library concentration was measured using a Qubit dsDNA High Sensitivity Kit (Thermo Fisher Scientific) and sequenced on the Illumina HiSeq 4000 platform.