From cryopreserved PBSC grafts of three donors, approximately 1x10^4 CD34+mono cells, 3x10^5 CD34-positive cells (CD34+cells, Lin-CD34highCD33-CD14-CD11b- cells), and 3x10^5 conventional monocytes (con-mono, Lin-CD34-CD33+CD14+CD11b+ cells) were directly sorted into tubes using FACSAriaTM II (BD Biosciences). After the collection of individual cell types, total RNA extraction was immediately performed with an RNeasyPlus Micro kit (QIAGEN) according to the manufacturer’s instructions. Thereafter, electrophoresis analyses were performed to examine the quality of the extracted RNAs using an Agilent 2100 Bioanalyzer (Agilent Technologies) with RNA Pico Chips (Agilent Technologies).
Label
biotin
Label protocol
Using 6ng of the extracted RNA in each sample with a GeneChip™ 3' IVT Pico Kit (Thermo Fisher Scientific), cDNA was made by reverse transcription, and cRNA was then synthesized and amplified by in vitro transcription. After the quality assessments, the cRNA was converted to biotinylated double-stranded cDNA hybridization targets.
Hybridization protocol
The product of cDNA was then hybridized into a human Clariom™ S Assay (Thermo Fisher Scientific) with a GeneChip™ Hybridization, Wash, and Stain Kit, and a GeneChip™ Hybridization Oven 645 (Thermo Fisher Scientific) via incubation at 45°C with rotation at 60 rpm for 16 hours.
Scan protocol
The arrays were automatically stained and read using a GeneChip™ Fluidics Station 450 and a GeneChip™ Scanner 3000 7G (Thermo Fisher Scientific), according to the protocol provided by the manufacturer.
Data processing
The raw data were analysed according to the algorithm of a Transcriptome Analysis Console (Thermo Fisher Scientific). affymetrix-algorithm-name = sst-rma-gene-full affymetrix-algorithm-version = 1.0 affymetrix-array-type = Clariom_S_Human program-name = Expression Console program-version = 1.4.1.46
