Encore complete rna seq dr multiplex system 1 8 and 9 16

RNA samples from 18 normal and 20 OA cartilage donors were sequenced using 150ng of total RNA as input. Sequencing mRNA libraries were prepared using the Encore Complete RNA-Seq DR Multiplex System 1–8 and 9–16 (NuGen, San Carlos, CA) with 16 unique indexed adapters (L2V6DR-BC2-L2V6DR-BC16). Two lanes of an Illumina HiSeq 2000 instrument were used to generate a total of 8–30 million 100bp reads.
The Illumina Genome Analyzer Pipeline Software (Casava v1.8.2) was used to convert the original image data generated by the sequencing machine into sequence data via base calling in order to generate fastq files and to demultiplex the samples. We performed a per base sequence quality check using the software FastQC (v0.10.1) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) prior to read mapping. Raw RNAseq reads were aligned to the human genome (hg19) using the STAR aligner [22 (link)]. The number of reads sequenced per sample ranged from 19–24 million reads, which should be sufficient for gene level quantification, but only 2–12 million reads per sample mapped to protein coding genes. To account for this issue, we applied high stringency the filtering of lowly expressed genes (log counts per million > 3) so that only the differential expression analysis included only genes that were expressed in high enough abundances to be confident in their relative gene expression values.

RNA samples were sequenced using 125–150ng of total RNA as input. Sequencing mRNA libraries were prepared using the Encore Complete RNA-Seq DR Multiplex System 1–8 and 9–16 (NuGen). RNA libraries were prepared using the Illumina TruSeq small RNA sample prep kit. Samples of each batch were pooled at equal amount of cDNA prior to sequencing, to control for lane and batch effects. Two lanes of an Illumina HiSeq 2000 instrument were used to generate a total of 8–30 million 100bp reads.
The Illumina Genome Analyzer Pipeline Software (Casava v1.8.2) was used to convert the original image data generated by the sequencing machine into sequence data via base calling in order to generate fastq files and to demultiplex the samples. We performed a per base sequence quality check using the software FastQC (v0.10.1) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) prior to read mapping. The number of reads sequenced ranged from 15–26 million reads per sample, with greater than 60% of these reads aligning to the human genome (hg19).