RNAseq
LYSIS
Laboratory correlative studies investigated possible biomarkers of response based on the transcriptome of peripheral blood CD34+ HSPCs. After ficoll centrifugation of blood, mononuclear cells were subjected to red blood cell lysis and CD34+ HSPCs were immunomagnetically isolated using the Ultrapure CD34 microbead kit (Miltenyi Biotec, Cat. No. 130-100-453) suing AUTOMACS (Miltenyi Biotec). RNA was extracted using Trizol (Invitrogen, Cat. No. 15596026). Briefly, the Smart-seq2 protocol was implemented with improved reverse transcription, template switching and preamplification to increase both yield and length of cDNA libraries generated from individual cells. The Illumina-compatible cDNA sequencing library was prepared with Nextera XT DNA Library Preparation Kit (Illumina, Cat. No. FC-131-1096). The quality, quantity, and the size distribution of the Illumina libraries were determined using the DNA-1000 Kit (Agilent Bioanalyzer, Cat. No. 5067-1504). Libraries were sequenced on the Novaseq 6000 (Illumina). Fastq files containing reads for each library were extracted and demultiplexed using Casava v1.8.2 pipeline. Sequencing adapter contaminations were removed from reads using cutadapt software tool (MIT) and the resulting reads were mapped and quantified on the transcriptome using RSEM v1.17For RNA-Seq of Differential expression analysis was performed using DESeq2 package