Rnase free disposable pellet pestle

Fish were euthanized by ice-chilled water immersion. After death, brain and retinal tissue were isolated through microdissection and homogenized using Fisherbrand™ RNase-free disposable pellet pestles. They were stored in liquid nitrogen until RNA extraction. Total RNA from brains and retinal tissue of age- and sex-matched wild-type, heterozygous, and homozygous Elovl4b KO fish was isolated using a Qiagen RNeasy mini kit following the manufacturer’s protocol. RNA was quantified using a NanoDrop spectrophotometer (Thermo Scientific). For the determination of mRNA expression levels and to validate germline mutagenesis, total RNA was reverse transcribed with a Verso cDNA Synthesis Kit (ThermoFisher) according to the manufacturer’s protocols using gene-targeting primers synthesized by the University of Utah DNA Peptide Synthesis core facility. The primer sets used are detailed in supplemental Table S1. The resultant RT-PCR products were resolved by gel electrophoresis on 2.5% agarose and imaged using an Invitrogen iBright™ CL750 Imager (Carlsbad, CA).

Tissues for assessing Lkb1 mRNA levels were flash-frozen immediately following harvest. While thawing in preparation for lysis, tissues were manually disrupted on dry ice using RNase-Free Disposable Pellet Pestles (Thermo Fisher Scientific:12-141-368). Tissues were repeatedly passed through a 20 G needle to yield a finer homogenate prior to the addition of RLT buffer containing 1% β-mercaptoethanol. RNA was extracted using Allprep DNA/RNA Mini Kit (Qiagen: 80204). cDNA was generated using the ProtoScript® First Strand cDNA Synthesis Kit (NEB: E6300). Measurements of Lkb1 and Gapdh expression levels were performed in triplicate using gene-specific primers (Lkb1 Fwd: 5’-CGAGGGATGTTGGAGTATGAG-3’; Lkb1 Rvs: 5’-AGCCAGAGGGTGTTTCTTC-3’; Gapdh Fwd: 5’-CAGCCTCGTCCCGTAGAC-3’; Gapdh Rvs: 5’-CATTGCTGACAATCTTGAGTGA-3’) and PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific: A25776) on an HT7900 Fast Real-Time PCR System with 384-Well Block Module (Applied Bioscience). Data were acquired using the Sequence Detection Systems Software v2.4.1 in Absolute Quantitation mode.

RNA extractions were conducted in parallel using a guanidinium thiocyanate-phenol-chloroform extraction method. Mosquito tissues were suspended in a 1 mL solution containing 4 M guanidine thiocyanate (CAS: 593-84-0), 0.5% Sarkosyl (CAS: 137-16-6), chloroform (CAS: 67-66-3), and 0.1 M 2-mercapthoethanol (CAS: 60-24-2). Using RNase-free disposable pellet pestles (Catalog #12-141-364, Thermo Fisher Scientific, Waltham, MA, USA) the samples are then manually homogenized until no visible structures remain. After tissue homogenization, the samples were extracted twice with phenol-chloroform. RNA was purified from the recovered aqueous solution using the RNAid Kit supplied by MPBio (catalog #111007200). RNAMATRIX beads were used to bind RNA using 5 μl for 5 whole heads and 10 μl for 120 sensory tissues. The beads were then washed twice using RNA Wash Concentrate to remove any remaining containments before eluting in 20 μl DEPC-treated water. Sample concentration and quality were determined using Thermo Scientific NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA).

Total RNA was extracted from the head kidney tissues using TRIzol (Invitrogen/Life Technologies, Carlsbad, CA, United States) following manufacturer instructions. The head kidney samples were lysed in TRIzol using RNase-Free Disposable Pellet Pestles (Thermo Fisher Scientific) before RNA extraction. The RNA samples (40 µg of each) were treated with 6.8 Kunitz units of DNase (Qiagen, Mississauga, ON, Canada) for 10 min at room temperature following the manufacturer’s instructions. The DNase-treated RNAs were then purified using the RNeasy MinElute Cleanup kit (Qiagen) based on manufacturer recommendations (Xue et al., 2015 (link); Caballero-Solares et al., 2017 (link)). The integrity and purity of purified RNA were checked using 1% agarose gel electrophoresis and NanoDrop spectrophotometry (NSW-1000), respectively. The RNA samples used in this study showed high purity (i.e., A260/280 and A260/230 ratios above 1.9 and 2.0, respectively) and integrity (tight 18S and 28S ribosomal RNA bands). One microgram of purified RNA was used for cDNA synthesis in a 20 µl reaction using random primers (250 ng; Invitrogen, Thermo Fisher Scientific), M-MLV reverse transcriptase (200 U; Invitrogen/Life Technologies), first-strand buffer (1× final concentration), dNTPs (0.5 mM final concentration), and DTT (10 mM final concentration) at 37°C for 50 min (Xue et al., 2015 (link)).

First, we selected one individual for each spider species, and dried the spider specimen using filter paper (Fig. 1a). Second, we transferred the dried specimen into a new 1.5 ml microcentrifuge tube (Thermo Fisher Scientific, USA), and grinded the sample into a fine powder using RNase-Free Disposable Pellet Pestles (Thermo Fisher Scientific, USA) on liquid nitrogen. Third, we added 900 μl Trizol (Invitrogen, USA) and 8 μl glycogen (Thermo Fisher Scientific, USA) to maximize the amount of pure RNA (Fig. 1a). Finally, we extracted total RNA from all selected individuals of 16 spider species following the Trizol manufacturer’s protocol (Fig. 1a).

Separate pint cages were set up to determine bacterial load of individual flies at specific time points (0 d, 3 d, 10 d, 14 d). Forty virgin males and forty virgin females were infected as previously stated for each treatment group. Once infected, individuals were given one hour to recover from anesthesia. After one hour, individuals that had not woken up were considered dead and removed. This point was also considered the start of the time course, and flies were aged to the appropriate time. Following incubation, flies were washed in 1X PBS, and placed into 100 µL of 1X PBS and homogenized using an RNase-free disposable pellet pestle (Fisher Scientific, 12-141-368, Waltham, MA) attached to a Pellet Pestle™ cordless motor (Fisher Scientific, 12-141-361, Waltham, MA). Following homogenization, the cultures were serial diluted and plated onto TSA for enumeration.