Truseq nano dna kit

Total RNA was isolated from FACS-sorted CD11b^highLy6G^high neutrophils using an RNAeasy Plus Micro kit (Qiagen). RNA integrity was verified by a Bioanalyzer (Agilent). Sample cDNAs were prepared using the SMART-Seq v4 Ultra Low Input kit (Takara) using 250 pg of input total RNA followed by library preparation using a TruSeq DNA Nano kit (Illumina). Libraries were verified by Tapestation (Agilent). Library concentrations were determined by real-time PCR with a StepOnePlus Real Time PCR System (Thermo Fisher) and a Kapa Library Quantification Kit (Kapa Biosystems/Roche). Libraries were sequenced with a 100 cycle single read protocol on a HiSeq 2500 (Illumina) with four libraries per lane. Fastq files were assembled using Bcl2Fastq2 (Illumina).

Samples were divided into males and females based on morphology and cleaned (as above) before RNA extraction. A third sample comprising all individuals including immature stages and eggs was sampled from within a single facial pore. RNA was extracted using a NucleoSpin RNA XS kit (Macherey-Nagel) for small tissue samples. cDNA synthesis was performed with the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories), with oligo(dT) priming. Library preparation was performed using the TruSeq DNA Nano kit (Illumina) with 350 bp inserts.

Total genomic DNAs of the three Iris species were extracted using the modified cetyltrimethylammonium bromide method [89 (link)]. Three genomic libraries were prepared using the TruSeq DNA Nano Kit (Illumina, San Diego, CA, USA) and sequenced using NextSeq500 platform (Illumina). A total of 3.2–3.4 gigabases (Gb) of paired-end reads (2 × 150 base pairs [bp]) were generated. Trimmed paired-end reads (Phred score ≥ 20) were assembled using the CLC genome assembler (version 4.06 beta; CLC Inc., Rarhus, Denmark) with the default parameters. SOAP de novo gap closer was used to fill in gaps based on alignments of paired-end reads [90 (link)]. Contigs were queried against the non-redundant database of the National Center for Biotechnology Information (NCBI) to identify those representing cp genomes, which were retrieved from the total contigs using Nucmer [91 (link)]. The aligned contigs were ordered using the cp genome sequences of I. koreana (NC_056174), I. minutoaurea (NC_056177), and I. odaesanensis (NC_056178) as references [12 (link)]. Finally, the trimmed paired-end reads were assembled into complete cp genome sequences using BWA software version 0.7.25 [92 (link)] (Figure S1). The newly sequenced chloroplast genomes in the present study were deposited in the NCBI GenBank database.

Libraries were prepared using TruSeq DNA Nano kit (Illumina Inc, San Diego, CA, USA), and samples were sequenced to an average depth of 30X using Illumina HiSeq X at the Core Genomics Lab at Sidra Medicine. Raw reads were aligned to GRCh37 using the standard settings of the BWA kit v0.7.15 [13 (link)]. Pre- and post-alignment quality checks were performed using FastQC v0.11.2 [14 ] and Picard v2.17.6 [15 ]. The heterozygosity and missingness rate were plotted after variant calling to evaluate the sample quality. We also performed a sex check, and we removed samples that shared a lot of variants, which implies contamination has occurred.

Whole blood was isolated from mice by cardiopuncture, and total RNA was isolated from FACS-sorted CD11b^highLy6G^high neutrophils using an RNAeasy Plus Micro kit (Qiagen). Integrity of RNA was verified by an Agilent Bioanalyzer prior to cDNA preparation using the SMART-Seq v4 Ultra Low Input kit (Takara). RNA libraries were prepared using a TruSeq DNA Nano kit (Illumina) and verified by Tapestation (Agilent). Library concentrations were measured by real-time PCR with StepOnePlus Real Time PCR System (ThermoFisher) followed by a Kapa Library Quantification Kit (KapaBiosystems/Roche). Libraries were then sequenced with a 100-cycle single-read protocol using a HiSeq 2500 sequencer (Illumina). Quality control checks were performed using the FastQC package, and raw reads were normalized using the DESeq2 Bioconductor package.