FLASH-seq of human and chimpanzee single spheroid budding at different time points
Human and chimpanzee spheroids budding at day 3 to day 6 of midgut/hindgut patterning were sorted manually into a 96-well format. Spheroids were lysed in a lysis buffer according to the original protocol of FLASH-seq. Reverse transcription and PCR amplification were carried out using SuperScript IV Reverse Transcriptase ( Thermo Fisher, 18090200) and KAPA HiFi HotStart ReadyMix (Roche, 07958935001). cDNA was purified using SeraMag SpeedBeads (Cytiva, 65152105050250) (0.8:1 bead-to-cDNA ratio) and quantified using the Quant-iT PicoGreen dsDNA assay (Thermo Fisher, P7589). Size distribution was assessed via an Agilent 2100 Bioanalyzer. Samples were normalized to 200 pg/Âµl for downstream library preparation.  Tagmentation was performed using homemade Tn5 transposase followed by heat inactivation with SDS. Enrichment PCR was conducted with KAPA HiFi DNA Polymerase (Roche, KK2502) and index adapters. 