Allprep dna rna mirna universal

RNA was extracted from CD138+ plasma cells using the AllPrep^® DNA/RNA/miRNA Universal or miRNeasy kits (Qiagen). RNA integrity was measured on an Agilent Bioanalyzer 2100 instrument; only samples with RNA integrity ≥7 were used for sequencing. Illumina-compatible RNA sequencing libraries were prepared using Scriptseq™ or Nextera technology and sequenced on Illumina HiSeq^® 1500 or 2500 instruments (Illumina). After pre-processing, filtered reads were aligned to the GRCh38 human reference genome using the STAR aligner tool.^{26 (link)} Gene read counts were normalised using the Reads Per Kilobase of transcript per million mapped reads (RPKM) method. In total, 51 annotated esterase genes (Supplementary Table S1) were identified in the human genome (assembly GRCh38) utilising the Ensembl release 99^{27 (link)} and NCBI^{28 (link)} databases, using the search term ‘esterase’ and further confirming the molecular function (gene ontology) of identified genes. A cut-off value of >1 RPKM was used to filter expressed esterase genes. DEseq2^{29 (link)} was used to identify variation in gene expression in newly diagnosed versus relapsed/refractory samples. The contribution of esterase gene expression to survival outcome was estimated by Kaplan–Meier analysis and performed using expression-based filtering. For each esterase gene, samples were stratified into ‘high’ (≥median expression) and ‘low’ (