Rnaqueous total rna isolation kit

RNA was isolated from preserved coral tissue samples from both in situ and ex situ samples using the RNAqueous Total RNA Isolation Kit (Invitrogen) according to manufacturer's recommendations. Coral tissue was removed from the skeleton using disposable blades and was then centrifuged to remove any remaining skeletal debris. After elution, RNA was DNased using 10 × DNase I (Thermo Fisher Scientific). First‐strand cDNA synthesis from a target of 1 μg DNased RNA per sample, cDNA amplification, barcoding, and sample pooling were performed according to protocols previously described (Lohman et al., 2016 (link); Wright et al., 2019 (link)). A total of 76 libraries were prepared from samples collected from the in situ LME and 12 libraries were prepared from samples in the deoxygenation challenge experiment (Table S1). Libraries from the in situ samples were sequenced on the HiSeq 2500 (Illumina) at the University of North Carolina (Chapel Hill, NC) and libraries from the deoxygenation challenge experiment samples were sequenced on the HiSeq 2500 (Illumina) at Tufts University Core Facility (Boston, MA). Sequenced reads have been uploaded to the National Center for Biotechnology Information Short Read Archive (SRA) under accession number PRJNA994102.

The biological and environmental samples were collected at local noon at 11-time points between 14 December 2011 and 10 January 2013 from the Isfjorden Adventfjorden time series station (IsA); located on the west coast of Spitsbergen, Svalbard (N 78°15.6, E 15°31.8, Fig. 1). At each of the 11 sampling dates, 30 l of seawater was sequentially sampled from 25 m depth using a 10 l Niskin bottle (KC Denmark), and immediately processed on board. Samples were kept in dark and cold conditions while prefiltered by gravity through 10 μm nylon mesh (KC Denmark) and then onto 8–12 47 mm 0.45 μm Durapore filters (Millipore) using vacuum pumps. Each filter was fixed in 600 μl LB buffer (RNAqueous Total RNA Isolation Kit, Invitrogen, Thermo Fisher Scientific) 5–20 min after sampling, flash-frozen in liquid nitrogen and stored at −80 °C.
At each sampling date, a vertical profile of environmental variables was obtained using a handheld SAIV 204 STD/CTD probe. However, in this paper we present only the data for 25 m depth where the samples were taken from (see^{10 (link)} for complete profile). Photosynthetically active radiation (PAR), size-fractionated chlorophyll a and nutrient concentrations (nitrate/nitrite, phosphate, silicate), were obtained as described in^{10 (link)}.

The longitudinal ability of CPMV to protect against CT26‐Luc challenge was carried out by incrementally increasing the time until CT26‐Luc i.p. tumor challenge. Mice were injected with 200 µg of CPMV and then with 500000 CT26‐Luc cells 7, 14, 21, and 28 days following CPMV injection.
To analyze CPMV biodistribution and clearance from the i.p. space, the i.p. fluid as well as the spleen, liver, kidneys, and lungs were collected after 1 and 2 weeks. Total RNA was extracted and purified from the solid organs using RNAqueous Total RNA Isolation Kit (Invitrogen) according to manufacturer's instructions. RNA from i.p. fluid was extracted using QIAamp Viral RNA mini kit (Qiagen) according to manufacturer's instructions.
Five nanograms of total RNA extracted from organs and i.p. fluid was probed for CPMV RNA using TaqMan Fast Virus 1‐Step Master Mix (Applied Biosystems). Serial dilutions (100, 10, 1, 0.1, and 0.01 ng CPMV RNA) were used as templates to graph a standard curve. All experiments included a no template control (NTC). CPMV Primers were designed and purchased from Integrated DNA Technologies:
Probe 5′‐/56FAM/TCGGGTTGT/ZEN/TGTTTGATGTTGGCC/3IABkFQ/−3′
Primer 1 (RV) 5′‐CAT GGA GTC TTG AGA GCA GAT AG‐3′
Primer 2 (FW) 5′‐ACA GCT ACC ACC AAC ATT TCT‐3′
This experiment was performed using a CFX96 touch real‐time PCR detection system (BioRad).

Total RNA was extracted from brain (supraesophageal) ganglia of the ventral nerve cord using RNAqueous^™ Total RNA Isolation Kit (Invitrogen, Cat. # AM1912), following the manufacturer’s instructions. Briefly, brain ganglia were dissected, and RNases inactivated by placing the tissue in RNA later Solution. The samples were homogenized in Lysis/Binding Solution (10–12 uL/mg), and an equal amount of 64% ethanol was added and mixed. The lysate/ethanol mixture was applied to a filter cartridge and centrifuged at 14,000 rpm. The filtered mixture was washed with 700 uL of Wash Solution #1, followed by washes with 2 x 500 uL of Wash Solution #2/3. RNA was eluted with 40–60 μL preheated Elution Solution, following another elution with a second 10–60 μL aliquot of Elution Solution. An Illumina Next generation Sequencing HiSeq2000 was employed for sequence analysis by the company MacroGen. The HiSeq2000 utilized HiSeq Control Software (HCS) v2.2.38 to generate raw images for system control and base calling through Real Time Analysis (RTA) v1.18.61.0, a software for integrated primary analysis. The base calls (BCL) binary was converted into FASTQ utilizing illumina package bcl2fastq (v1.8.4).

Total RNA was purified from tissues according to the manufacturer's instructions using RNAqueous total RNA isolation kit (Invitrogen AM1912). Additional steps were conducted to inactivate DNases. cDNA was then synthesized from the resulting RNA using oligo(dT)20 and Super-Script III (Invitrogen 18080051).
Genes were quantified by real-time PCR using SYBR Green primers (Table S2; MilliporeSigma) and was conducted on Applied Biosystems Step One Plus. Data analysis was carried out using log twofold change normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression.