Single cell RNA-seq to resolve heterogeneity in a cardiac differentiation timecourse
Single cell RNA-seq to resolve heterogeneity in a cardiac differentiation timecourse
Differentiations were started on sequential days so the different timepoints could be collected and processed on the same day. Sample 1 was collected on day 3 and day 3.75 (50:50 mix); Samples 2, 3 and 4 were collected on days 4.75, 5.75 and 12 respectively. Embryoid bodies were dissociated to single cells using TrypLE Select.  Gene expression libraries were prepared from the single cells using the Chromium Controller and Single Cell 3ʹ Reagent Kits v3 (10x Genomics, Inc. Pleasanton, USA) according to the manufacturer's protocol (CG000183 Rev A). Briefly, nanoliter-scale Gel Beads-in-emulsion (GEMs) were generated by combining barcoded Gel Beads, a master mix containing cells, and partitioning oil onto a Chromium chip. Cells were delivered at a limiting dilution, such that the majority (90-99%) of generated GEMs contain no cell, while the remainder largely contain a single cell. The Gel Beads were then dissolved, primers released, and any co-partitioned cells lysed. Primers containing an Illumina TruSeq Read 1 sequencing primer, a 16-nucleotide 10x Barcode, a 10-nucleotide unique molecular identifier (UMI) and a 30-nucleotide poly(dT) sequence were then mixed with the cell lysate and a master mix containing reverse transcription (RT) reagents. Incubation of the GEMs then yielded barcoded cDNA from poly-adenylated mRNA. Following incubation, GEMs were broken, and pooled fractions recovered. First-strand cDNA was then purified from the post GEM-RT reaction mixture using silane magnetic beads and amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were then used to optimize the cDNA amplicon size. Illumina P5 & P7 sequences, a sample index, and TruSeq Read 2 sequence were added via end repair, A-tailing, adaptor ligation, and PCR to yield final Illumina-compatible sequencing libraries.