Fig. 3

Applying SCAPTURE pipeline to study PASs in human PBMC datasets. a Application of SCAPTURE to identify exonic PASs from 3′ tag-based scRNA-seq datasets. Left, statistics of called peaks and identified PASs in six PBMC scRNA-seq datasets from 10x Genomics. Right, comparison of SCAPTURE-identified exonic PASs with known exonic PASs. Overlapped or non-overlapped exonic PASs identified by SCAPTURE were analyzed with known PAS annotation shown in Fig. 2a. b Nucleotide distribution of sequences at known (left), overlapped (middle), and non-overlapped (right) exonic PASs. Upstream (–) 100 bp to downstream (+) 100 bp sequences of PASs were analyzed. c Validation of SCAPTURE-identified non-overlapped PASs. Four such cases (salmon rectangle) in the NADK, PPP2R5C, MTX1, and SMIM11B gene loci highlighted with their polyadenylation signals (AAUAAA motif) and further designed for validation by PCR as previously reported [26]. A SCAPTURE-identified PAS that was previously annotated in the NADK gene locus (purple rectangle) was shown as a positive control. Top panel, TruSeq RNA-seq (gray), Smart-seq2 (dark blue), and 10x Chromium (rose) scRNA-seq from published datasets [17]. Middle panel, wiggle tracks from one of 10x Genomics PBMC scRNA-seq datasets (the 10 k PBMC dataset) were indicated. Bottom panel, Ribo–RNA-seq datasets of bulk cells from normal (gray) and SLE patient (dark red) PBMC samples [25]. PCR primers were shown in the bottom for experimental validation. See “Methods” section for details. Asterisk, non-specific RNA-seq signals due to multiple mapping. d Validation of SCAPTURE-identified non-overlapped exonic PASs. Corresponding PCR products of these non-overlapped exonic PASs (salmon arrow) were shown with correct sizes. One overlapped exonic PAS (purple arrow) that was also identified by SCAPTURE in the NADK gene locus was shown as the positive control. As negative controls, no PCR products from downstream regions of these SCAPTURE-identified non-overlapped PASs were detected. Mixed PBMC RNA samples from SLE patients previously examined by Liu et al. [25] were used for this validation. Of note, additional nested primers were designed to validate the SCAPTURE-identified PAS in the PPP2R5C locus. e Comparison of signature poly(A) signal motifs from exonic PASs identified by SCAPTURE or by two other recently reported pipelines, Sierra [28] and scAPA [29]. f Nucleotide distribution of sequences around exonic PASs identified by SCAPTURE, Sierra, or scAPA in six PBMC datasets from 10x Genomics. Upstream (–) 100 bp to downstream (+) 100 bp sequences of PASs were analyzed