RNA-Seq of human: H7 Day 1 PPS
hPSC-derived cells or primary cord blood HSPCs were either directly lysed in the culture plate, or purified via FACS as indicated and then lysed, using 350 L of RLT Plus Buffer and RNA was extracted using the RNeasy Plus Micro Kit (Qiagen) according to the manufacturers protocol. RNA concentration and quality was assessed by Agilent Bioanalyzer using the High Sensitivity RNA Analysis kit (Agilent, 5067-1513), to ensure that RNA was of high quality (RNA integrity number [RIN] > 7). 1 ng of total RNA was further processed to synthesize cDNA using the SMART-Seq2 pipeline (Picelli et al., 2014). cDNA size distribution and concentration were assessed by Agilent Bioanalyzer using the High Sensitivity DNA Analysis kit (Agilent, 5067-4626). The Illumina Nextera XT DNA Sample Preparation kit (Illumina FC-131-1096) was used for library construction and the KAPA Library Quantification Kit (Kapa Biosystems, KK4854) was used for library quantification. Library size distribution was assessed by Agilent Bioanalyzer. RNA-sequencing libraries were sequenced on the Illumina NovaSeq 6000 sequencer, to obtain 2x150bp paired-end reads (PE150). Kallisto (Bray et al., 2016) was used to align reads to the hg38 human reference genome (Homo_sapiens.GRCh38.96.gtf, downloaded from Ensemble) and to quantify transcript level abundances. Default parameters were used for quantification, and data were imported into DESeq2 (Love et al., 2014) using tximport (Soneson et al., 2015) for all downstream analysis in R, including differential gene expression analysis.