Extended Data Fig. 1: Most of the intronic reads arise due to internal priming from stable positions.

a–d, Examples of read density around intronic polyA and polyT sequences. The browser screenshots show the density of reads from the 10x Chromium mouse hippocampus dataset (top track of each panel), mouse bone marrow inDrop dataset (second track from the top), and chromaffin differentiation assessed using SMART-seq2 (third track). The bottom two tracks show gene annotation, and positions of polyA or polyT sequences with a length of at least 15 bp with one allowed mismatch. The polyA/polyT boxes are coloured blue if the stretch is in a concordant orientation to the transcription of the underlying gene (that is, would result in a polyA sequence in the nascent RNA molecule being transcribed), or red if they are oriented in the discordant position (that is, would result in a polyT sequence in the RNA). The 3′-end-based 10x Chromium and inDrop protocols show discrete peaks downstream of the polyA priming sites, with the 10x dataset also showing peaks upstream of the polyT sites. The SMART-seq2 protocol shows much more diffused peaks, expected from the full-length purification procedure used by the protocol. e–h, Average read density profiles around concordant and discordant internal priming sites. The plots show observed/expected intronic read density around (A)₁₅ or (T)₁₅ sequences (with 1 allowed mismatch) within the intronic regions. The x axis shows position relative to the motif position (in basepairs), in a genomic reference orientation. The bold lines show genome-wide average (trimmed of two extreme values among chromosomes for each position). The averages of individual chromosomes are shown as semi-transparent lines. e, Profiles of mouse hippocampus 10x Chromium dataset (n = 18,213). f, Profile for human forebrain 10x data (n = 1,720). g, Profile for the chromaffin differentiation data measured using SMART-seq2 (n = 385). h, Profile for the mouse bone marrow data measured using inDrop (n = 3,018). The top left corner of each plot shows the number of all intronic reads (that is, falling within the gene, but not touching an exon) that falls within the 250 bp around internal priming sites (1,500 bp was used for the SMART-seq2 dataset). In 10x data, while concordant internal priming sites produce stronger signal, their frequency within the genome is lower than those of discordant sites, so that overall discordant sites account for slightly higher fraction of intronic signals. By contrast, the inDrop dataset appears to have very limited discordant priming.