Illumina HiSeq 2500 paired end sequencing; Single-cell RNA-seq of the mouse developing heart (pilot study)
Single-cell RNA-seq of the mouse developing heart (pilot study)
Embryos of the appropriate stage were dissected in fresh M2 medium (Sigma-Aldrich), keeping the yolk sac intact. Embryos were considered to be at the LHT stage once both sides of the cardiac crescent were completely folded and fused. Linear heart tubes were micro-dissected using tungsten needles, keeping the overlying endoderm intact. Samples were dissociated into single-cells using 200μl Accutase (ThermoFisher) for 12 minutes at 37C, being agitated every 2 minutes before adding 200μl heat-inactivated FBS (ThermoFisher) to quench the reaction. Cells were then centrifuged at 1000rpm for 3 minutes at 40C before being suspended in 100μl HBSS (ThermoFisher) + 1% FBS and stored on ice. Single-cells were collected using a Sony SH800 FACS machine using a stringent single-cell collection protocol and sorted into 384 well plates containing SMART-seq2 lysis buffer 31 plus ERCC spike-ins at a concentration of 1:10million. To ensure good quality cells were collected a live/dead dye 438 (Abcam) was used; 100μl was added to the cell suspension at a 2x concentration in HBSS 10 minutes before collection, live cells were collected based on their FITC intensity. Once cells were collected, plates were sealed, spun down, and frozen using dry ice before being stored at -80C.  There was no presequencing visual checks on the sorted cells. QC was performed on the sequencing data to exclude poor quality libraries:any samples with fewer than 50,000 total reads mapped to exons; less than 6,000 detected genes; more than 30% of reads mapped to ERCC spike-ins; or more than 15% of reads mapped to mitochondrial genes were excluded from the dataset. mRNA from single cells was isolated and amplified (25 PCR cycles) using the SMART-seq2 protocol. Multiplexed sequencing libraries were generated from cDNA using the Illumina Nextera XT protocol.