RNA sequencing comparing the effect of the introduction of RUNX1-D171N (RHD) in control- or SCN -CSF3R-d715 hematopoietic progenitor cells (HPCs)

To address how CSF3R and RUNX1 mutations affect hematopoiesis in a severe congenital neutropenia (SCN) background we used SCN patient, or control, derived induced pluripotent stem cells (iPSC). HPCs were generated from CRISPR-Cas9 genome edited CSF3R-d715 SCN or control-derived iPSCs with the STEMdiff™ Hematopoietic Kit (STEMCELL Technologies). The RUNX1-D171N (RHD) mutation, or an empty vector control, was lentivirally introduced to the hematopoietic induction cultures 60 hours 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 HiSeq 2500 or Novaseq 6000 (both Illumina).