Single stranded cDNA was mixed with Affymetrix hybridization controls (GeneChip Hybridization Control Kit, Affymetrix) and hybridized on Affymetrix GeneChip Human Gene ST 1.0 array (Affymetrix) utilizing GeneChip Hybridization, Wash, and Stain Kit (Affymetrix) according to manufacturer protocol. Arrays were hybridized in Affymetrix Hybridization Oven 645 and washed / stained / labeled in Affymetrix GeneChip Fluidics Station 450.
Affymetrix CEL files were normalized using RMA algorithm at the gene level with default settings (general background correction, quantile normalization and median polish summarization) in Affymetrix Expression Console software. Further analyses were performed in GeneSpring and/or R-script and or Affymetrix PowerTools. Please note that 40 identifiers in the normalised data file cannot be found in the Reporter Name column of A-AFFY-141. Instead, they can be found in the Comment[transcript_cluster_id] column of A-AFFY-187, which is the same Affymetrix array design but with slightly more updated annotation. The 40 idnetifiers involved are stored in an additional file (E-MTAB-1038.additional.1.zip) which can be found under Browse all available files link.
Arrays were scanned by Affymetrix GeneChip Scanner 3000 operated by Affymetrix GeneChip Command Console (AGCC) software and DAT files were stored. Grid of each array was manualy realigned and finally the CEL files were generated.
Maximum input 250ng total RNA was mixed with appropriate amount of Affymetrix Poly-A controls (GeneChip Eukaryotic Poly-A RNA Control Kit, Affymetrix) and further processed by Ambion WT assay (Life Technologies) according to manufacturer protocol. This WT assay workflow comprises of total RNA amplification (in vitro transcription) and cRNA purification, and single stranded cDNA reverse transcription and its purification.
Total of 5.5ug was fragmented and labeled using GeneChip WT Terminal Labeling Kit (Affymetrix) according to manufacturer protocol.
Total RNA from CD138+ cells was isolated using QIAGEN RNeasy Mini Kit according to manufacturer protocol. Concentration and purity of isolated total RNA was measured by NanoDrop spectrophotometer (Thermo Scientific). RNA integrity number (RIN) was measured by Agilent 2010 Bionalyzer (Agilent Technologies). Total RNA with 260/280 absorbance ratio >1.7 and RNA integrity number (RIN) >7.5 followed further processing.
The bone marrow of multiple myeloma patients was obtained during routine clinical procedure. Plasma cells in mononuclear cell fraction were enriched by anti-CD138+ immunomagnetic beads and sorted using AutoMACS (Miltenyi Biotec) (magnetic activated cell sorting). Purity of CD138+ fraction was measured by flowcytometry and/or cytospin slides. Samples with >80% CD138 positive cells followed further processing.