Figure 7. Comparative analysis of ScriptSeq and DLAF libraries prepared from mouse embryonic cortices (ECx).

(a) Base frequency in the genomic sequences upstream of read_1 in ScriptSeq libraries. Data are averaged from three biological replicates. The sequence shows a clear bias towards GATCT, which is similar to a part of template-switching oligo. (b) Coverage along the length of transcripts. RNA-SeQC coverage for each percentile of gene length is shown for the 5,000 middle-expressed genes. The coverage is normalized to the total number of reads mapping to the 5,000 middle-expressed genes in each library. (c) Distribution of the first-sequenced nucleotides of read_1. The first bases are plotted across the TSSs. Yellow (DLAF) and red (ScriptSeq) lines are 5-base moving average. DLAF read_1 but not ScriptSeq shows a peak around +1, 0 and −1 positions. In b and c, dashed and solid lines denote individual and averaged replicates obtained from 5,000 middle-expressed genes. (d–f) Comparison to DeepCAGE data. Read_1-start positions of ScriptSeq and DLAF are shown for Actb (d). Malat1 (e) and Actg1 (f) loci. CAGE data are derived from the cerebellum (Cbl), embryo (Emb) and hippocampus (Hip)¹³. DLAF but not ScriptSeq peaks largely match with CAGE signals. DLAF libraries treated with Klenow show decreased and broader signals downstream of TSSs in a dose-dependent manner. Coverage is normalized to the total non-rRNA and non-mtRNA reads for the dUTP and DLAF libraries.