Rnalater

Selected BM CD34+/lin- cells of 80 CP-CML patients were resuspended in 50 μl of RNAlater (Thermo Fisher Scientific, Milano, Italy) and stored at -20°C until RNA extraction was performed as previously described [18 (link)].
Total RNA was isolated from the BM CD34+/lin- cells stored in RNAlater using MagMAX 96 Total RNA Isolation Kit (Thermo Fisher Scientific) [18 (link)], according to the manufacturer’s instructions. The quality and the yield of the extracted RNA were measured using Nanodrop (Thermo Fisher Scientific) (see http://dx.doi.org/10.17504/protocols.io.yncfvaw).

The inferior conjunctival cytology was fixed in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for 24 h. Then, RNAlater was removed and the samples were stored at −80 °C until being processed.
The RNA isolation and purification of the samples were performed with the commercial kits QIAshredder and RNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions.
Twenty-two µL of the total RNA were used for the first-strand cDNA synthesis that was performed with the High Capability cDNA Reverse Transcription Kit and random hexamer primers (Thermo Fisher Scientific). The quantitative PCR was carried with the QuantStudio 3 system (Thermo Fisher Scientific) by using the cDNA, Quantitect SYBR Green Kit (Qiagen), and specific primers of IL-1β, IL-6, and MMP9 (Table 2). The hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1) gene was used as an internal control to normalize the mRNA relative expression. Each sample was triplicated and negative controls were included in all the measurements. The thermal cycler program was as follows: 15 min at 95 °C, 40 cycles of 15 s at 94 °C, 30 s at 55 °C, and 34 s at 72 °C.
The analysis of the melting curves confirmed the specificity of the primers and the absence of primer-dimers. Finally, both the stability of the HPRT1 gene and the analysis of the quantitative PCR data were performed by the 2^−ΔCt method.

Upon sampling, fish were euthanized with benzocaine (800 mg/L). Blood was collected from the caudal vein in heparinized tubes (Kruuse ltd UK). Heparinized blood was centrifuged (130 g for 10 min) and plasma and blood cells were separated. Blood was used for Western blot (WB) and plasma for assessing specific antibody. An aliquot of heparinized blood was centrifuged (12 000 g for 5 min) in glass microhematocrit tubes (Vitrex Medical A/S) with specific centrifuge (Nüve) and haematocrit (hct) assessed visually with specific scale.
The heart was cut in two equal halves along the midsagittal axis; one half was stored in 10% neutral-buffered formalin for histopathological evaluation, and the other half was divided into two aliquots: one was stored in RNALater^® (ThermoFisher Scientific Inc, USA) for gene expression analysis (CD4 and CD8) and one was stored in RLT buffer (© QIAGEN) for quantifying viral RNA. The spleen was divided into three aliquots: one was stored in 10% neutral-buffered formalin, one was stored in RNALater^® (ThermoFisher Scientific Inc,) for gene expression analysis and one was stored in RLT buffer (QIAGEN) for quantifying viral RNA. Gill, liver, pancreas and pyloric caeca, distal intestine, red and white muscle, mid kidney were also collected and stored in 10% neutral-buffered formalin for histopathological evaluation.

Microbial mats were collected at the Mariana back-arc sites Snail (also known as the Fryer Site) and Urashima and at the Mariana Arc sites NW Eifuku and NW Rota-1 (Figure 1A) during R/V Roger Revelle cruise 1413 (11/29/2014–12/21/2014) with remotely operated vehicle Jason II. A total of 22 samples were collected with either the biomat syringe sampler (Breier et al., 2012 (link); Figure 1F) or scoop sampler (Figure 1G, Table 1). Scoop samples were preserved in RNAlater (ThermoFisher Scientific, Waltham, MA) at depth (LSc), or RNAlater was added after the scoop was brought to the surface (Sc). All mats collected were stored at −80°C upon processing until DNA extractions. Sample names consist of Jason II dive number (797–801) followed by biomat sampler cassette letter and individual syringe number(s) or the type of scoop and the scoop number (e.g., biomat sampler: 797B3 and scoop sampler: 797LSc1). Mats from three locations were collected with both biomat and scoop samplers to compare community structure and diversity in relation to sampling technique. Vent effluent was collected with the Hydrothermal Fluid and Particle Sampler (Butterfield et al., 2004 ) and analyzed on board for total hydrogen sulfide, pH, and dissolved hydrogen. Iron was measured by atomic absorption at Pacific Marine Environmental Lab in Seattle, WA.