Nextera library preparation kit

Given the minimal amount of RNA obtained from IVLE, the Smart-Seq2 protocol was adapted for low-input RNA library preparation (25 (link)). Two different amounts of input RNA, 12 ng and 3 ng, for each sample and its replicates were prepared. Polyadenylated mRNA was enriched using 5 mg/ml Dynabead Oligo(dT)₂₀ in 1X PBS (pH 7.4) from mRNA DIRECT kit (Thermo Fisher). Beads were washed with 10mM Tris-HCl pH 7.5, 150 mM LiCl, 1mM EDTA pH 8.0, 0.1% w/v LiDS in nuclease free water and RNA eluted using 10 mM Tris-HCl pH 8.0 at 75°C for 2 minutes. The poly A-enriched RNA was reverse transcribed by Smart-Seq2 as described (25 (link)) with 10 reverse transcriptase cycles, and cDNA further amplified using IsoSeq PCR (ISPCR) primers with 10 or 11 PCR cycles. Dual indexed sequencing libraries were made out of 5 ng cDNA from the above preparations using Illumina Nextera library preparation kit according to manufacturer’s instructions (Illumina). Quality checked and equimolar pooled libraries were sequenced in a HiSeq 4000 Illumina system and 75bp paired- non-stranded- reads generated. Sequence data were deposited in the European Nucleotide Archive (ENA) with the study number ERP128933; NCBI BioProject ID PRJEB44842 (accession numbers for each sample are shown in Supplementary Table S1).

Metagenomic libraries were prepared with the Nextera library preparation kit (Illumina) using 1 to 10 ng of input DNA. After library generation, 12 to 16 uniquely barcoded libraries were pooled and run on an Illumina NextSeq 550 platform using a NextSeq 500/550 mid-output kit (Illumina) set to 150-bp single-end reads, which yielded averages of 10 million and 7.3 million reads from positive and negative samples, respectively. After removal of reads mapping to human DNA, averages of 1.7 million (positive) and 1.9 million (negative) microbial reads were used for further analysis.

Library was prepared using the Nextera library preparation kit (Illumina, Carlsbad, CA) following the manufacturer's protocol. Library was quantified with Picogreen, and 20 µl of 2.5 nM diluted DNA was used for library preparation. Library was denatured and diluted to 20 pM following the protocol recommended for sequencing of Nextera libraries on the MiSeq sequencer (Illumina). Sequencing was performed on the MiSeq system using paired-end reads of 150 nucleotides. Genomes were assembled using Ray 2.0.0 [56] (link). Reads were aligned to L. infantum JPCM5 version 4 using the bwa aligner [57] (link), and sequencing coverage was assessed for each position of chromosome 6. The sequencing data for the FCPO amplicon are available at the EMBL European Nucleotide Archive (http://www.ebi.ac.uk/ena) under the study accession number ERP002431, sample accession ERS227354.

Total RNA purified from mTEC^lo (Figure 1—figure supplement 1) was extracted with miRNeasy Micro Kit (QIAGEN), and RNA quality was assessed on an Agilent 2100 BioAnalyzer (Agilent Technologies). RNA Integrity Number values over 8 were obtained. RNA-seq libraries were generated using the SMART-Seq-v4-Ultra Low Input RNA Kit (Clontech) combined to the Nextera library preparation kit (Illumina) following the manufacturer’s instructions. Libraries were sequenced with the Illumina NextSeq 500 machine to generate datasets of single-end 75 bp reads. Two independent biological replicates were used per each condition. RNA-seq data have been deposited with Gene Expression Omnibus (GEO) under the accession number GSE144650.

Metagenomic libraries were prepared with the Nextera library preparation kit (Illumina) using 1 to 10 ng of input DNA. After library generation, 12 to 16 uniquely barcoded libraries were pooled and run on an Illumina NextSeq 550 platform using a NextSeq 500/550 mid-output kit (Illumina) set to 150-bp single-end reads, which yielded averages of 10 million and 7.3 million reads from positive and negative samples, respectively. After removal of reads mapping to human DNA, averages of 1.7 million (positive) and 1.9 million (negative) microbial reads were used for further analysis.

Mouse genomic DNA was PCR amplified using primer sets described in Supplementary Table 4. DNA library was prepared using the Nextera library preparation kit (Illumina). The concentrations of the indexed libraries were analyzed on the Agilent 2200 TapeStation using the D1000 Kit (Agilent Technologies). Equimolar amounts of the indexed libraries were pooled to obtain a 4 nM library mixture. After denaturing and further diluting, the final 12 pM library was loaded into an Illumina cartridge. Sequencing was performed using the Illumina MiSeq Reagent Kit v2 (500 Cycles) on the Illumina MiSeq instrument following the manufacturer’s instructions.