Affymetrix genechip wt terminal labeling and controls kit

CBSC and rNK or aNK cells were co-cultured for 4 h and then stained with anti-CD34-PE (BD Biosciences) for re-isolation of CBSC (as CD34⁺ cells) by sorting using a FACSAria cell sorter (BD Biosciences). RNA was isolated from sorted CBSC, rNK or aNK cells using the RNeasy Micro kit (Qiagen). Samples were processed using GeneChip Whole Transcript Sense Target Labeling assays using the Ambion WT Expression kit and Affymetrix GeneChip WT Terminal Labeling and Controls kit (Affymetrix). The resulting ssDNAs were hybridized to the GeneChip human Gene 2.0 ST Array (Affymetrix) and microarray analysis was performed by the UCL Genomics Facility, Institute of Child Health (London, UK). Image reads were processed using Affymetrix software and background was corrected and normalized using the RMA algorithm with GeneSpring software (Agilent Technologies). Differentially expressed genes were analyzed using GeneSpring software. Data are available from the EMBL-EBI/ArrayExpress repository under accession E-MTAB-2531 for the CBSC analysis and E-MTAB-2847 for the NK cell analysis.

The frozen oral mucosa samples were homogenized with TRIzol^R Reagent (Invitrogen, ThermoFischer Scientific), and total RNA was isolated with the mirVana^TM miRNA Isolation Kit (Ambion/Invitrogen, ThermoFischer Scientific) according to the manufacturer’s protocol. RNA amplification was performed with the Ambion^R WT Expression Kit (Applied Biosystems, ThermoFischer Scientific), according to the manufacturer’s instructions, starting with 100 ng total RNA, on a TP Basic Thermocycler, real time PCR instrument (Biometra, Göttingen, Germany). The quality of the RNA product was evaluated on the NanoDrop spectrophotometer and the 2100 Bioanalyzer with the Agilent RNA 6000 Nano Kit (Agilent Technologies, Santa Clara, CA, US). We prepared the RNA samples for hybridization to Affymetrix GeneChip Human Exon 1.0 ST Arrays with the Affymetrix GeneChip WT Terminal Labeling and Controls Kit (P/N 901524) (Affymetrix, Santa Clara, CA, US), according to the manufacturer’s instructions. CEL files were generated with Affymetrix GeneChip Command Console Software and deposited at the NCBI Gene Expression Omnibus repository, under number GSE81979. A similar procedure was applied to analyze gene expression in MNCs isolated from blood samples.