Labchip gx touch

The RNA sequencing was performed by BIKEN Biomics services (Osaka, Japan). Small RNAs were extracted from 200μl plasma samples with a miRNeasy Serum/Plasma Kit (Qiagen) in accordance with the manufacturer’s protocol. The small RNA libraries were prepared from 6 μl of the RNA sample using an NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB) according to the manufacturer’s instructions. The concentration and size distribution of the libraries was measured by a Qubit (Thremo Fisher Scientific) and LabChip GX Touch (Perkin Elmer) and the size selection was performed by electrophoresis with 2% E-Gel EX Agarose Gel (Thremo Fisher Scientific) to trim the primer-dimer products. Sequencing was performed on an Illumina MiSeq platform in a 50-base single-end mode. The sequencing reads were mapped to a human miRNA reference sequence and analyzed by using miRbase (http://ccb.jhu.edu/software/tophat/index.shtml) and CLC genomics workbench v9.5.3 software (Qiagen). After trimming under default parameter settings to retain only reads with lengths 15–25 bp, the annotated miRNAs were normalized by measuring endogenous cel-miR-39 that was spiked in all samples at the same concentration (5 fmol) and then calculated as read counts per million mapped reads (RPM). MiRNAs with read counts more than 10 were analyzed for further data analysis.

The bacterial community composition associated with algal cultures was determined by 16S metabarcoding. A mock community comprising a mix of DNA from 26 cultivated bacterial strains (Thomas et al., 2019 (link)) and negative control were run and treated in parallel to the DNA extracts. For all of these samples, the V3 and V4 regions of the 16S rDNA gene were amplified using the NOCHL primers including Illumina adapters (Thomas et al., 2019 (link)), to avoid plastid DNA amplification. Then a standard Illumina protocol for metabarcoding (Illumina, 2013 ) was run using the Q5® High-Fidelity PCR Kit (New England BioLabs, MA, USA), the AMPure XP for PCR Purification Kit (Beckman Coulter, Brea, CA, USA), and the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA). Libraries were quantified with a Quantifluor® ds DNA System (Promega, WI, USA), and mean fragment size was determined using a LabChip® GX Touch™ (Perkin Elmer, MA, USA). An equimolar pool of all samples was generated at a concentration of 4 nM, diluted to 3 pM, spiked with 10% PhiX (Illumina), and sequenced on an Illumina MiSeq sequencer at the Genomer platform (Station Biologique de Roscoff) using a MiSeq v3 kit (2x300bp, paired-end). Raw Illumina reads were deposited at the European Nucleotide Archive under project accession number PRJEB47035.

DNA was normalized at 0.2 ng/μL in order to start with 1 ng to perform a sequencing library preparation using Nextera XT kit (Illumina) following the supplier's instructions. A final AMPure beads purification at ratio 0.6 was performed on a Sciclone robotic platform from Perkin Elmer. The quality and quantity of each library were evaluated using a capillary electrophoresis method (LabChip GX Touch from Perkin Elmer). Libraries were pooled based on molarity calculated by the LabChip GX Touch. The equimolar pool was assembled using a Hamilton robotic platform.

RNA was extracted from ankle joints and quadriceps using 1 ml and 0.5 ml respectively of TRIzol™ reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. The quality of the RNA was assessed on a LabChip GX touch (Perkin Elmer) and quantified using the Promega QuantiFluor RNA system® as per instructions. Gene expression analysis of RNA was performed using the commercially available NanoString™ nCounter® mouse Myeloid Innate Immunity gene expression panel (NanoString™ Technologies, Seattle, WA, USA) following the manufacturer’s instructions. This panel contains 20 internal reference genes for data normalisation and 754 target genes including several known to be regulated during CHIKV infection. Raw gene expression data was normalised against a set of positive and negative controls to account for background noise and platform associated variation. Reference gene normalisation was performed using the GeNorm Algorithm where housekeeping genes were selected based on the lowest variance across samples.

Frozen tissue was ground into a fine powder using a ⅛′′ steel ball bearing with 1 min shaking at 25 Hz in a TissueLyser II (Qiagen, Hilden, Germany). Total RNA was extracted from finely ground tissue using TRIzol reagent (#T9424‐200ML; Sigma‐Aldrich, St. Louis, MO, USA) at a ratio of 1 ml solution per 100 mg ground tissue. Residual phenol was removed from the crude extract through two chloroform extractions at a ratio of 1:5 (v/v), followed by precipitation using isopropanol at 1:1 (v/v). The precipitated RNA was washed twice with 70% ethanol and resuspended in 1 mm sodium citrate buffer (pH 5.4). RNA quantification was performed through spectrophotometric analysis at 260 nm using the Nanodrop ND‐1000 Spectrophotometer, and RNA quality was assessed using 1% agarose gel electrophoresis or the LabChip GX Touch (PerkinElmer, Waltham, MA, USA). Five micrograms of purified total RNA was combined with 5 μl of TURBO DNase buffer and 1 μl TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) in a 50 μl reaction and incubated at 37°C for 30 min. DNA nuclease‐treated RNA was then purified using 1.8× Sera‐Mag paramagnetic particles.

10x Genomics single-cell immune profiling technology was used for single-cell sequencing of antigen-specific B cells. Single-cell suspensions were loaded onto the GEM channels of the Chromium Controller microfluidics device (10x Genomics) and processed following the manufacturer’s protocol for target cell recovery of at most 10,000 B cells. Single-cell 5′ RNA sequencing (RNA-seq) and BCR sequencing (BCR-seq) libraries were prepared using the Chromium Next GEM Single Cell 5′ Kit v2 (10x Genomics) and the Chromium Single Cell Human BCR Amplification Kit (10x Genomics), respectively. The antigen, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq), and hashtag antibody libraries were prepared and amplified using a 5′ Feature Barcode Kit (10x Genomics). All library preparations were performed according to the manufacturer’s protocols, and the final libraries were assessed using a LabChip GX Touch (PerkinElmer) instrument to verify the cDNA fragment size. The 5′ RNA-seq, BCR-seq, and feature barcode libraries were analyzed, quantified, and sequenced using DNBSEQ-G400 (MGI). This resulted in approximately 50,000 reads per cell for each 5′ RNA-seq library.