Agilent 2200 tapestation system

Total RNA concentration and RNA integrity (RNA integrity number equivalent; RINe) from liver tissue were measured using a Qubit 2.0 fluorometer (Thermo Fisher Scientific, Tokyo, Japan) and an Agilent TapeStation 2200 System (Agilent Technologies, Tokyo, Japan). High-quality RNA samples were used for RNA-Seq analyses. The RNA-Seq analyses were performed by Eurofins Genomics (Eurofins Genomics, Tokyo, Japan) with eight groups of RNA samples (glucose and L-fucose oral administration group, four samples per group, diluted to 5.5 μg/55 μL) using a HiSeq2500 system (Illumina, San Diego, CA, USA). The original sequence reads were cleaned and mapped to the mouse genome gene reference (GRCm38.mm10) using QIAGEN’s CLC Genomics Workbench v.11.0.1 (https://digitalinsights.qiagen.com/). Differentially expressed genes (DEGs) were identified between the glucose and L-fucose oral administration group using the reads per kilobase per million mapped reads (RPKM) values of corresponding transcripts. The filtering standard was set to more than a 1.5-fold change, and FDR p-value ≤ 0.05. The DEGs containing up- and downregulated genes were used for gene ontology (GO)-term analysis using the Database for Annotation, Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf.gov/) tools [23 (link),24 (link)] for their functional classification.

Given logistic and sampling limitations, only four samples of P. ventilabrum from Langenuen/Korsfjord were used for the transcriptomic analysis (Table 1, with asterisks): two females and two nonreproductive specimens. The protocols used for RNA isolation, mRNA purification and cDNA library preparation were described in Koutsouveli et al.^{94 (link)}. Briefly, total RNA extraction was conducted with a standard TRIzol™ Reagent (ThermoFisher Scientific) protocol, according to the guidelines of the manufacturer. Further mRNA purification was performed with the Dynabeads mRNA DIRECT kit (ThermoFisher Scientific), applying the final stage of the protocol, ‘Elimination of rRNA contamination’. The quantity and quality of mRNA were assessed by NanoDrop 2000 (ThermoFisher Scientific). Then, cDNA libraries were prepared with Scriptseq v2 kit (Illumina) (according to the manufacturer’s instructions), using an initial mRNA quantity of 50 ng. The amount of cDNA was then assessed with Qubit™ dsDNA HS Assay kit (ThermoFisher Scientific) and the quality with an Agilent Tapestation 2200 system (Agilent Technologies). The sequencing was done in an Illumina NextSeq 500 platform at the Natural History Museum of London sequencing facility (Molecular Core Labs).

For the DNA extraction, mycelial samples were harvested and freeze dried for 24 h. The total DNA was isolated from the freeze-dried samples using the Wizard^® Genomic DNA Purification System (Promega, Madison, WI, USA) as per the manufacturer’s instructions to achieve high-molecular-weight DNA. The eluted DNA was quantified using a Qubit^TM dsDNA BR Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), and the quality was assessed using a Nanodrop 2000 (Thermo Fisher Scientific). The integrity and molecular weight were assessed using an Agilent TapeStation 2200 system (Agilent Technologies, Santa Clara, CA, USA) following manufacturer’s instructions.
For RNA extraction, mycelial samples were collected into 1.5 mL Eppendorf tubes and stored at −80 °C until they were processed. The RNA extraction was carried out using a Plant/Fungi Total RNA Purification Kit (Norgen Biotek Corp., Thorold, ON, Canada). Quality was assessed using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific), and integrity was assessed using an Agilent TapeStation 2200 system.

Total RNA was isolated from placental tissue using an miRNeasy Mini Kit (Qiagen, Valencia, CA, USA). RNA was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The integrity of the RNA was assessed on an Agilent TapeStation 2200 system (Agilent Technologies, Santa Clara, CA, USA). Purified RNA aliquots were stored at −80 °C until further processing. Genomic DNA was obtained from placental tissue using the standard extraction protocol and stored at −30 °C until use.

Before the DNA extraction, the devices containing resuspended F and CWF samples were mixed by gentle inversions at least 3–4 times to further homogenate the contents of the entire container. 500 uL of F and CWF samples were recovered and used for the DNA extraction carried out by the FastDNA™ Spin Kit for Soil (MP Biomedicals, Santa Ana, CA, USA), according to the manufacturer instructions. A 40 s bead-beating step at speed 6 was executed on the FastPrep Instrument (BIO 101, Carlsbad, Canada). The DNA was eluted in a 100-μL volume of sterile water and stored at -20 °C. DNA quality was evaluated using the Agilent TapeStation 2200 System (Agilent, Santa Clara, CA, USA) with Genomic DNA ScreenTape assay (Agilent, Santa Clara, CA, USA). The DNA Integrity Number (DIN) was assigned to each sample by applying Agilent 2200 TapeStation software (controller version A.01.05). Then, quantitative fluorimetric analysis was carried out using Quant-iTTM PicoGreen® dsDNA Assay Kit (Invitrogen, Carlsbad, California) on a NanoDrop 3300 Fluorospectrometer (Thermo Fisher Scientific, Waltham, MA, USA).

Quality of the DNA was checked on 0.8% Agarose (A9539, Sigma-Aldrich, St. Louis, MO, USA) gel at 120 V for approximately 60 min or until the samples reached 3/4th of the gel. Absorbance ratio at 260/280 was measured with a NanoDrop 2000 UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). A Qubit 2.0 Fluorometer (Q32866, Invitrogen, Carlsbad, CA, USA) was used with a Qubit dsDNA HS Assay Kit (Q32854) to confirm DNA input of 10 μg before shearing. All the DNA samples passed the QC were subjected to paired-end sequencing library preparation with NEB Ultra DNA library preparation kit (New England BioLabs, Ipswich, MA, USA). The quantity and quality check of library was carried out using Agilent TapeStation 2200 System (Agilent Technologies, Santa Clara, CA, USA). Whole genome sequencing of the four eucalypt samples was performed by AgriGenome Labs Pvt Ltd, Hyderabad on an Illumina HiSeq 4000/X ten Genome Analyzer using 2 × 150 bp chemistry. The fastq files were pre-processed using AdapterRemoval2 (v2.2, default parameters). The raw reads were checked for presence of adapter sequences and reads that’s average quality score less than 30 (<30 phred score) in any of the paired-end reads were filtered out.

The endometrial tissues were divided into three groups (n = 9 for each) according to Kenney and Doig’s categories: I, IIA, and IIB² . Total RNA was isolated from around 60 mg of endometrial tissue and homogenized with the use of TriReagent® (T9424; Sigma-Aldrich) and controlled in terms of quality using the Agilent TapeStation2200 system. Within each group, every three RNA isolates were pooled in equimolar ratios into one sample to obtain three replicates (pools). The pooling of samples was performed to reduce the experimental costs and the variability among individual samples and at the same time have more individuals within an experiment.
RNA integrity number (RIN) was assessed for each RNA isolate using Agilent 2100 system and Expert software (Agilent Technologies, Inc., Santa Clara, CA, USA). Only samples with a RIN above 8.0 were processed further. In total, 500 ng of RNA was used for library construction with the TruSeq RNA Sample Prep v2 kit (Illumina, San Diego, CA).