Wild type hiPSC cell KOLF2.C1 were seeded in 6-well plate at 50k cells per well and then differentiated in different conditions (BS, BSA, BSA-83). Differentiated cells were harvested at day 6 for total RNA extraction using NucleoSpin® RNA Plus (Takara, #740984.250) according to the instructions.  For time course bulk RNA sequencing.  WT and HAND1-/- KOLF2.C1 were seeded in 6-well plate as 50k cells per well and then cultured in StemFlex media or differentiated at different time points to ensure collection of differentiated cells at the same day. RNA-seq libraries were prepared with KAPA Stranded Total RNA-Seq kit (Roche) according to manufacturer's instruction. The samples were normalized to 300ngs input and spiked in with ERCC. First, ribosomal RNA was depleted using RiboErase followed by DNase treatment. Purified RNA was then fragmented at 85°C for 6 mins, targeting fragments range 250-300bp. Fragmented RNA is reverse transcribed with an incubation of 25°C for 10mins, 42°C for 15mins and an inactivation step at 70°C for 15mins. This was followed by second strand synthesis at 16°C, 60mins. Double stranded cDNA fragments were purified using Ampure XP beads (Beckman). The dscDNA were then A-tailed, and ligated with illumina unique adaptors (illumina). Adaptor-ligated DNA was purified using Ampure XP beads. This is followed by 10 cycles of PCR amplification. The final library was cleaned up using AMpure XP beads. Quantification of libraries were performed using real-time qPCR (Thermo Fisher). Sequencing was performed on Illumina NovaSeq platform generating paired end reads of 2X150bp.