RNA-Seq of CD138-positive cells purified from bone marrow samples

Bulk RNA-Sequencing of CD138-positive cells (plasma cells) purified from bone marrow samples of patients with MGUS, asymptomatic myeloma (AMM) and multiple myeloma (before treatment (MM) and at relapse (MMR)).RNA-seq is performed using the protocol optimised for low input analysis of CD138-purified plasma cells. The extracted total RNA (5 ng, minimum 10 pg) is used to generate the full-length double-stranded cDNA. For this, first-strand cDNA is synthesised, applying the SMARTer Ultra Low RNA Kit (Clontech laboratories, Illumina), and purified using SPRI AMPure XP Beads (Beckman Coulter). Subsequently, the double-stranded cDNA is amplified by long-distance PCR and again purified using SPRI AMPure XP Beads (Beckman Coulter). The Agilent 2100 BioAnalyzer and the Agilent High Sensitivity DNA Kit (Agilent Technologies) are used to quantify and validate the purification. Full-length cDNA is randomly sheared into smaller fragments using the Covaris system, and 10 ng are used for library preparation, according to the Illumina Sequencing protocol (New England BioLabs) and using the NEBNext ChIP-Seq Library Prep Master Mix Set. Sequencing is performed with 2x50-bp or 2x75-bp paired-end unstranded RNA-seq on an Illumina HiSeq2000.