RNA Samples Used for RNA-Seq Libraries
Libraries were made from RNA extracted from two human cell lines, the A549 lung cancer line and the 293f kidney carcinoma cell line. Total RNA was isolated using BiooPure™ RNA Extraction Reagent (PerkinElmer) and RNA concentration was determined by Nanodrop analysis of UV absorbance. Some of the RNA samples were processed to select polyadenylated transcripts as described below. Prior to poly A selection, 1 µL of a 1:10 dilution of ERCC controls (precisely quantified synthetic polyadenylated transcripts that do not share sequence homology with human sequences) was added to the RNA samples. Use of the ERCC control RNAs allowed assessment of the extent to which the relative levels of the different ERCC control transcripts observed in the RNA-Seq results correlated with the known relative levels of the ERCC transcripts in the control mixture.
Triplicate barcoded strand-specific, directional RNA-Seq libraries were made using NEXTFLEX® Poly(A) Bead selected input RNA and sequenced on the Illumina® HiSeq® platform. The libraries were made with indexing adapters (“barcodes”) to allow them to be pooled for multiplex sequencing. Poly(A) selection was carried out using 5 µg of input total RNA and two rounds of selection with various amounts of PerkinElmer’s NEXTFLEX® Poly(A) Beads (20 µL, 50 µL , 100 µL ).
The RNA-Seq libraries from NEXTFLEX® Poly(A) Bead selected input RNA and total RNA were produced from 35 ng input RNA using the NEXTFLEX® Rapid Directional RNA-Seq Kit. The input RNA was chemically fragmented to an average size of 300 bases using the reagents and protocol included in the kit. Libraries were amplified using 12 cycles of PCR. Yields and size distribution of libraries were assessed on an Agilent® Bioanalyzer®. Equal amounts of the barcoded libraries were pooled prior to sequencing and sequenced in a single flow cell on an Illumina® HiSeq® instrument. After applying standard quality filters, results were analyzed to determine the extent of residual rRNA contamination in each library. Analysis was also carried out to determine the relative levels of ERCC controls and of reads mapping to different categories of endogenous transcripts.
The yields of poly(A)+ RNA ranged from ~50 ng – 80 ng, corresponding to ~1%-2% recovery. These values are in good agreement with accepted values for the proportion of total RNA that is comprised of mRNA. Representative results from Bioanalyzer® analysis of the libraries made using poly(A)-selected RNA are shown in Fig 1 below. The Bioanalyzer® data showed high yields and narrow size distribution of the products, consistent with high-quality RNA libraries.
Figure 1. Directional RNA-Seq library from RNA selected using 20 µL of NEXTFLEX® Poly(A) Beads. The concentration of the library was 80 nM.
Comparison of rRNA Contamination Using NEXTFLEX® Poly(A) Bead-Selected and Total RNA
Table 1 shows the extent of rRNA contamination in the libraries prepared from total RNA and the dramatic reduction in contaminating rRNA in the NEXTFLEX® Poly(A) Bead selected samples.
Table 1. RNA-Seq Reads Mapping to Ribosomal RNA
Relative Levels of ERCC Control RNAs in RNA-Seq Libraries Made from NEXTFLEX® Poly(A) Bead Selected and Total RNA
Figure 2. Correlation between expected and observed values for percentage of reads mapping to 94 ERCC control RNAs in RNA-Seq library made using 20 µL of NEXTFLEX® Poly(A) Beads.
Relative Levels of Different Categories of Endogenous Transcripts Detected in RNA-Seq Libraries Made from NEXTFLEX® Poly(A) Bead Selected and Total RNA
Figure 3. Percentage of reads mapping to rRNA regions of interest with and without Poly(A) bead selection.
PerkinElmer’s NEXTFLEX® Poly(A) Beads are an effective method to eliminate most sequencing reads that map to ribosomal RNA while retaining faithful representation of relative levels of polyadenylated RNAs. When total RNA was used as input for library construction, the vast majority of reads mapped to rRNA, whereas only a small percentage of reads mapped to rRNA in the libraries made using RNA selected using NEXTFLEX® Poly(A) Beads. The reduction in reads mapping to rRNA in the libraries prepared using NEXTFLEX® Poly(A) Bead selection is associated with a corresponding increase in reads mapping to exons and other categories of interest. Analysis of observed reads mapping to the various ERCC control RNAs, compared to the known relative levels of these RNAs that were spiked into the sample prior to poly(A) selection, demonstrates excellent correlation over a wide range of abundance of the different synthetic transcripts. The NEXTFLEX® Poly(A) Beads used in conjunction with the NEXTFLEX® Rapid Directional RNA-Seq Kit provide a straightforward workflow for generating high-quality RNA-Seq data, facilitating the discovery of new biological relationships that are reflected by global analysis of complex transcriptomes.