Single cell RNA-seq to resolve heterogeneity in a cardiac differentiation timecourse from wild-type and HAND1-null hESCs
Single cell RNA-seq to resolve heterogeneity in a cardiac differentiation timecourse from wild-type and HAND1-null hESCs
Embryoid bodies were dissociated to single cells as described above. Cells from different conditions were combined as follows: 25% SB-treated cells, 25% DMH1-treated cells and 50% control (vehicle-only) cells. Samples were collected at day 3, 4, 5, 6, 7, 8 and 10. Additional wild-type replicates at days 7 and 8 were included. A total of 1-2 x 106 cells were used for each sample. The Single Cell Fixed RNA Sample Preparation Kit (10X Genomics) was used to fix and cryopreserve the cells according to the manufacturer's instructions.  The Single Cell Fixed RNA Sample Preparation Kit (10X Genomics) was used to fix and cryopreserve the cells according to the manufacturer's instructions. In brief, cells were fixed with Fixation Buffer containing 4% formaldehyde (ThermoFisher Scientific) overnight at 4°C. 24 hours later, cells were quenched with freshly prepared quench buffer and stored at -80°C. Frozen cells were thawed and washed in 0.5X PBS/0.02% BSA (Miltenyi)/0.2 U/ml RNase Inhibitor (Roche).  Barcoding was performed using the Chromium Next GEM Single Cell Fixed RNA Human Transcriptome Probe Kit (10X Genomics) according to the manufacturer's instructions. Up to 2 x 10^6 cells per timepoint and genotype (wild-type and HAND1-null) were re-suspended in Hybridisation Mix with a single Human WTA Probe (10x Genomics, Inc. Pleasanton, USA) according to the manufacturer's protocol (CG000527 Rev C). Following GEM generation, a ligation step sealed the nick between the left- and right-hand probe and subsequently the Gel Bead primer was hybridised to the capture sequence on the ligated probe pair and extended by a polymerase adding the UMI, 10x Barcode and partial Read 1 sequence before a heat-denaturation step inactivated the enzymes. Post-incubation, GEMs were broken and the recovered ligated product pre-amplified before being cleaned up by SPRIselect. Illumina-compatible sequencing libraries were constructed by adding P5, P7, i5 and i7 sample indexes, and Illumina Small Read 2 sequences to the 10x barcoded, ligated probe products via Sample Index PCR followed by a SPRIselect size-selection.