Supplementary Figure 6: Validation of mRNA 3′ end sequencing using Quant-seq.

(a) Quant-seq uses total RNA as input, hence no prior poly(A) enrichment or rRNA depletion is required. Library generation is initiated by oligo-dT-anchor priming. Primer already contains Illumina-compatible linker sequences (green). After first-strand synthesis, the RNA is removed and second-strand synthesis is initiated by random priming and a DNA polymerase. The random primer also contains Illumina-compatible linker sequences (shown in blue). No purification is required between first and second strand synthesis. The insert size is optimized for shorter reads (SR50 or SR100). Second strand synthesis is followed by a magnetic bead-based purification step. The library is then amplified, introducing the sequences required for cluster generation (shown in red and purple). External barcodes (BC) for multiplexing are introduced during the PCR amplification step. (b) Normalized coverage of poly(A)-selected RNA-seq (top three panels) and Quant-seq (bottom three panels) generated from mESCs and analyzed across 24.440 Entrez genes. Coverage is reported for 500 bp regions surrounding the 5′ end or the 3′ end of all genes. Gene body coverage was normalized to gene length (including introns). (c) Gene expression values determined by Quant-seq correlate with RNA-seq-derived, transcript-length-normalized expression in mESCs. Length normalized RNA-seq signal (in TPM) is compared to gene-tag values (counts per million, CPM) for 17.821 genes. Average of three independent cell culture replicates is shown. Spearman correlation coefficient (r_S) and associated p-Value is indicated. (d) Quant-seq produces highly-reproducible gene expression values. Comparison of 20.818 transcripts from three independent biological Quant-seq replicates (r#1-r#3) generated from total RNA of mESCs are shown. Spearman correlation coefficient (r_S) and associated p-Value are indicated.