RNA sequencing comparing SCN CSF3R-d715 RUNX1-D171N (RHD) treated with the HDAC inhibitor MS275, octyl-(R)-2hydroxyglutarate (2HG) or solvent control (DMSO)

Effects of TET2 on repressing inflammation have been assigned to its methylcytosine dioxygenase activity, but also to its ability to recruit HDAC-mediated repressor activity to pro-inflammatory genes. To discriminate between the two above-proposed functions of TET2, we asked how inhibition of its enzymatic activity, by administration of octyl-(R)-2hydroxyglutarate (2HG), and its ability to recruit HDAC-activity, by administration of the HDAC-inhibitor MS275 (Entinostat), affected the transcriptional profile of CSF3R-d715 RUNX1-D171N (RHD) expressing SCN-iPSC derived CD34+CD45+ HPCs. HPCs were generated from CRISPR-Cas9 genome edited CSF3R-d715 SCN-derived iPSCs with the STEMdiff™ Hematopoietic Kit (STEMCELL Technologies). The RUNX1-D171N (RHD) mutation was lentivirally introduced to the hematopoietic induction cultures 60 hours before harvesting the floating cells. DMSO (solvent control), 0.1 mM Octyl-(R)-2HG (Sigma-Aldrich) or 2 µM MS275 (Santa Cruz) was added for 16 hours to the hematopoietic induction cultures before harvesting the floating cells. Floating cells were harvested at Day 12 of the hematopoietic induction protocol. CD34+CD45+ HPCs were FACS sorted in TRIzol and RNA was isolated according to the manufacturer’s protocol. SMARTer Ultra Low Input RNA kit for sequencing (Clontech, v4 Cat# 634891) was used to generate cDNA. Sequencing libraries were generated using TruSeq Nano DNA Sample Preparation kits (Illumina, Cat# 20015964), according to the low sample protocol and paired-end sequenced on a Novaseq 6000 (Illumina).