Single cell 3’ transcriptome profiling

Abstract

Whole 3’ transcriptome profiling at the single cell level opens up new abilities for researchers to answer complex questions. Thousands of individual cells per sample are Barcoded separately to index the transcriptome of each cell individually. It is done by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where cells are delivered at a limiting dilution, such that the majority (~90-99%) of generated GEMs contain no cell. The 16 bp 10x Barcode and 12 bp UMI are encoded in Read 1, while the poly(dT) primers are used in this protocol for generating Single Cell 3’ Gene Expression libraries. After GEM generation, copartitioned cells are lysed and reverse transcription (RT) was performed after which all cDNA from single cell share a common Barcode. Full-length cDNA was amplified via PCR to generate sufficient mass for library construction. This is followed by enzymatic fragmentation and size selection to optimize the cDNA amplicon size. Library construction was finished via End Repair, A-tailing, Adaptor Ligation, and PCR. P5, P7, i7 and i5 sample index, and TruSeq Read 2 (read 2 primer sequence) were added. TruSeq Read 1 and TruSeq Read 2 are standard Illumina sequencing primer sites used in paired-end sequencing. The library prepared in this way, containing the P5 and P7 primers, is ready for Illumina amplification.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202