Truseq rnaseq sample prep kit

The RNAseq libraries were prepared from cDNA averaging 230 nt using Illumina’s TruSeq RNAseq Sample Prep kit. The four transcriptome libraries were pooled in equimolar concentration, quantified by qPCR, and sequenced from both ends of the fragment (100 bp reads) using a TruSeq SBS sequencing kit (version 3). Two lanes were sequenced on a HiSeq2000 at the University of Illinois, Urbana-Champaign, Il, USA. The data were analyzed with Casava 1.8 (pipeline 1.8)
[94 ], which also filtered out low-quality reads. Adaptor sequences were also removed from the dataset. The sequencing yielded ca. 314 M read-pairs from the four transcriptomes and for the two lanes in total.

The extracted mitochondrial RNA from the flower buds (3–5 mm size) in CMS line 2074A, its maintainer 2074B and fertile material F₁ (2074A × E5903) were sequenced on an Illumina HiSeq2000 at BGI (Beijing Genomics Institute). Ribosomal RNAs were removed from the extracted mitochondrial RNA using Ribo-Zero (Epicentre, Madison, WI) and the mitochondrial RNA libraries were prepared using Illumina’s TruSeq RNAseq Sample Prep kit. Libraries were sequenced on one lane with 4 Gb clean reads/samples of an average length of 90 nt for paired-end. RNA sequence data quality was checked using FastQC to remove the adapters, low-quality, containing N bases and short sequences with reads Q30 > 85%. The reads were mapped to the assembled mitogenome of CMS line 2074A using bowtie2 [54 (link)] with the following parameters: -D 5 -R 1 -N 0 -L 25 -i S, 1, 2.00. Then, the resulting SAM files from bowtie2 mapping were transformed into BAM files using samtools view program [45 (link)]. The bowtie2 mapping results of these pair-end reads in BAM files were then used to calculate read count for each gene through HTSeq-count program [55 (link)]. Differentially expressed genes that showed up and down regulation between samples were defined based on the standards of cutoff: two-fold change and a p-value of less than 0.05.

RNA quality was assessed at UIUC with Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). We prepared RNA-seq libraries with unique barcodes using Illumina’s TruSeq RNA-seq Sample Prep kit. Libraries were quantified by qPCR and pooled before sequencing. We sequenced the pooled libraries on eight lanes for 101 cycles from each end using a TruSeq SBS sequencing kit (version 3) on an Illumina HiSeq2500. We used Casava 1.8.2. to generate Fastq files.
For the DNA sequencing we extracted DNA from leg and wing tissues of four bees using the MOBIO power soil DNA extraction kit (MOBIO Laboratories (now Qiagen), Carlsbad, CA). Shotgun genomic libraries were prepared using the Kapa Hyper-Prep kit (Kapa Biosystems Inc., Wilmington, MA). Libraries were quantified by qPCR and sequenced for 161 cycles from each end on an Illumina HiSeq2500 using TruSeq sequencing kit version 1. We generated and demultiplexed Fastq files with the bcl2fastq v1.8.4 Conversion Software (Illumina, San Diego, CA).