Single-cell polyadenylation sequencing (scPolyA-seq) reveals the dynamic of polyA site usage during cell cycles in tumor cell lines [C1]

Alternative polyadenylation (APA) plays an important role in post-transcriptional gene regulation. But how genes' APA usages are influenced by cell cycle remains largely unknown. Here we developed single cell polyadenylation sequencing (scPolyA-seq), a strand-specific approach for sequencing 3' end of transcripts which can get very deep sequencing level around polyA site for each cell, to investigate the landscape of APA at single cell level, based on Fluidigm C1. We compared APA changes before and after thymidine double blocking (TdR) synchronization of HeLa cells. We then identify 6 gene clusters whose distal polyA site usages fluctuate during cell cycle. Overall design: We performed scPolyA-seq, a single-cell RNA sequencing method we devised based on fluidigm C1, on cell line mixtures of Mouse embryonic fibroblasts (MEFs) + synchronized HeLa and MDA-MB-468 + HeLa. We used a Fluidigm C1 HT IFC chip, which could separate 800 single cells, including 20 columns and 40 rows. Cell mixture1 (suspensions of MEFs containing ~10% synchronized HeLa cells, column 1 to 10) and cell mixture 2 (suspensions of MDA-MB-468 containing ~10% HeLa cells, column 11 to 20) were loaded into two independent inlets of a C1 HT integrated fluidics circuit (IFC), thus up of 400 cells could be captured for each cell suspension. Totally 16 columns passed library quality control and were sequenced in PE 150bp. Read 1 was only used as cell barcode, while Read 2 was used in further analysis. Each column of data would be then demultiplexed into 40 cells by a Perl script named “mRNASeqHT_demultiplex.pl” according to the cell barcode in Read 1. Two bulk libraries of MDA-MB-468 cells were also constructed using the same protocol. This series includes mouse and human cell lines, but we mainly focus on analyzing human cell lines in our paper. Notice: Read 1 carries cell barcode, while Read 2 may contain polyA sites and sequences nearby. Read 2 is used for identifying the polyA sites and calculating gene expression in our work. As some researchers want to re-use our Read 1 data, we also upload these raw data with '_R1' in their file names, but users must be cautious that sequence quality may be quite low after polyT region, which is common issue for NGS.