Nextera xt dna library prep

1 ng of SMART-Seq v4 amplified cDNA (Takara Biosystems 634,890) was used for Nextera XT DNA library prep (Illumina, FC-131-1024). For sample and input specifications see Table 2.
Libraries were sequenced using a NextSeq 500 (Illumina). Quality control on fastq files was performed with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
Reads were aligned to Danio rerio reference genome GRCz10 with STAR v2.4.2a using a two-pass strategy. Genes were quantified using the Danio_rerio.GRCz10.91.gtf transcriptome.
The DESeq2 R-package (version 1.20.0) was used for count normalization and differential gene expression analysis. The normalized read counts were used to generate PCA plots, heatmaps and the correlation matrix. Pearson’s correlation coefficient was calculated on log transformed normalized read counts. Pre-ranked Gene Set Enrichment Analysis (GSEA) was preformed using GenePattern 2.0 (https://cloud.genepattern.org/gp/pages/login.jsf).

At 12 weeks of age, mice (n = 9/genotype) were single housed for 2 weeks and colon content was collected into collection tubes containing DNA stabilization buffer. Whole metagenome shallow shotgun sequencing (at least 2 million paired-end reads/sample) was performed using the Illumina miSeq or Illumina NextSeq instrument (the instrument used is dependent on the number of samples in a batch, Illumina, San Diego, CA). For the analysis, samples were extracted using the Qiagen PowerMag Microbiome DNA Isolation kit (Hilden, Germany) on the King Fisher automated platform (Thermo Scientific, Waltham, MA). Isolated DNA was quantitated using a fluorescent concentration assay and normalized to prepare for library prep using the Illumina Nextera XT DNA Library prep recommendations. Runs were spiked with 1% PhiX. Standard processing used 2 x 150 base pair paired-end sequencing with dual 8 base pair indexes. The instrument run takes ~29 hours. Criteria for acceptable results: final run must have a cluster density of 180-230K/mm² with greater than 80% of clusters passing filter, and at least 75% of bases must call at a minimum Phred score of Q30 (99.5%).

Two sequencing libraries were generated from each single cell cDNA preparation, one for the TRA/TRB repertoire and one for the 5′ ends of transcripts, and, subsequently, sequenced as described previously [39 (link)]. Briefly, for transcriptome analysis, 5′ end transcriptome libraries were extended from the purified cDNA by a 12 cycle PCR using the Nextera XT DNA library prep (Illumina) and sequenced according to the Illumina TruSeq Rapid v2 protocol on Illumina HiSeq2500 sequencer. Single reads were generated of 50 bp in length with a single 8 bp index sequence. For TRA/TRB profiling, TRA and TRB transcripts were amplified using specific PCR reactions which also added the Illumina adapter sequences. From the resulting TR sequencing libraries, paired-end reads of 300 bp in length were generated with an 8 bp index on an Illumina MiSeq sequencer per the manufacturer’s instructions (Illumina, San Diego, CA, USA).

For transcriptome analysis, part of the purified cDNA was tagmented using the Nextera XT DNA library prep (Illumina) and Illumina adaptor and indices sequences were extended by a 12 cycle PCR according to the manufacturer's instructions (Figure 1B). The resulting whole transcriptome sequencing libraries were purified and size-selected for ~400–900 bp fragments with AMPure XP beads. The sequencing libraries were quantified with the Quant-it assay and checked on a Bioanalyzer using the High sensitivity DNA kit. Sequencing libraries were sequenced according to the Illumina TruSeq Rapid v2 protocol on Illumina HiSeq2500 sequencer. Single reads were generated of 50 bp in length with a single 8 bp index sequence.

Cofactor Genomics performed quality control on RNA samples, and RNA integrity was determined using a the Agilent 2100 Bioanalyzer. Samples with RNA integrity numbers between 8 and 10 were used for library construction. Total RNA was reverse-transcribed using an Oligo (dT) primer (Co-Factor), and limited cDNA amplification was performed using the SMARTer Ultra Low Input RNA Kit for Sequencing–v4 (Takara Bio USA, Inc). Full-length cDNA was fragmented and tagged, followed by limited polymerase chain reaction enrichment to generate the final cDNA sequencing library (Nextera XT DNA Library Prep; Illumina). Libraries were sequenced as single-end 75–base pair reads using an Illumina NextSeq500 following the manufacturer's instructions. Because the amount of nasal epithelium in each sample was very limited, we were not able to perform confirmatory quantitative polymerase chain reaction.

Standard methods were used for isolation of chromosomal DNA [54 ]. One nanogram of genomic DNA was used for library generation by the Nextera XT DNA Library Prep (Illumina). Sequencing was carried out on a MiSeq benchtop sequencer and performed in paired-end modus (2 × 300 bp) using a MiSeq Reagent Kit v3 cartridge (600-cycle kit). Reads were mapped against the 1701 targets of the L. monocytogenes core genome MLST scheme [27 (link)], using the Ridom SeqSphere Software (Münster, Germany). Sequence types (STs) and CTs were determined after automated allele submission to the cgMLST server for L. monocytogenes (http://www.cgmlst.org/ncs/schema/690488/). Minimum spanning trees were calculated in the ‘pairwise ignore missing values’ mode.

Total RNA was processed for library construction by Cofactor Genomics (St. Louis, MO, USA) according to the following procedure. Briefly, total RNA was reverse-transcribed using an Oligo(dT) primer, and limited cDNA amplification was performed using the SMARTer Ultra Low Input RNA Kit for Sequencing—v4 (Takara Bio, Shiga, Japan). The resulting full-length cDNA was fragmented and tagged, followed by limited PCR enrichment to generate the final cDNA sequencing library (Nextera® XT DNA Library Prep, Illumina, San Diego, CA, USA). Libraries were sequenced as single-end 75 base pair reads on the Illumina NextSeq500 per the manufacturer’s instructions.