GSM8078801: Amphioxus_21hpf_scRNAseq S2; Branchiostoma lanceolatum; RNA-Seq
MARS-seq was performed as described in Keren-Shaul et al. 2019 (10.1038/s41596-019-0164-4). All 40 plates (a total of 15,384 single cell libraries) were prepared using the same conditions and reagents. Using a Bravo automated liquid handling platform (Agilent), mRNA was reverse transcribed into cDNA using the lysis oligonucleotide containing unique molecular identifiers (UMIs) and cell barcodes. Residual, unused oligonucleotides were removed by treatment with Exonuclease I (NEB). cDNAs were pooled and linearly amplified using T7 in vitro transcription. The resulting RNA was fragmented and ligated to an oligo containing the pool barcode and Illumina sequences, using T4 ssDNA:RNA ligase. RNA was reverse transcribed into DNA and PCR-amplified with 14 cycles. 21hpf embyos (19C) were dissociated and pooled before sorting single-cells for scRNAseq or ATAC-seq experiments.