RNA extraction, sequencing and transcriptomic analyses
LYSED, HEAT, CLL
Sample banking was conducted at centralised labs (University Hospitals Cologne, Kiel and Ulm, Germany, and Labcorp, NC, USA). CD19-positive cells (i.e. CLL cells) were enriched from peripheral blood mononuclear cells (PBMC) using CD19-positive microbeads following the manufacturer’s instruction (Miltenyi Biotec # 130-050-301). The median B-cell fraction of samples prior to CD19-enrichment, as assessed via immunophenotyping, was 97%. CD19-positive cells were lysed in RLT buffer and total RNA was isolated using RNeasy Mini Kit (QiAgen #74106). Thereby, RNA molecules longer than 200 nucleotides were enriched and eluted with nuclease-free water. RNA integrity (RIN) was measured by Agilent Bioanalyzer and accepted samples only passed QC. Sequencing libraries were created using Illumina TruSeq Standard mRNA method, which preferentially selects for messenger RNA (mRNA) by taking advantage of the polyadenylated tail. Briefly, total RNA samples were concentration normalized, and poly-adenylated RNA was purified using oligo-dT attached to magnetic beads. The purified mRNA was fragmented using heat in the presence of divalent cations and converted into double-stranded cDNA. They underwent end-repair, A-tailing, and polymerase chain reaction (PCR), after which they were quantified, normalized and pooled in preparation for sequencing. Libraries were sequenced using the Illumina sequencing-by-synthesis platform, with a sequencing protocol of 50 bp paired-end sequencing and total read depth of 50 M reads per sample.RNA sequencing data in FASTQ were analysed using HTSeqGenie (v3.16.1) in BioConductor (v3.0) as follows: first, reads with low nucleotide qualities (70% of bases with quality <23) or matches to rRNA and adapter sequences were removed. The remaining reads were aligned to the human reference genome (human: GRCh38.p10) using GSNAP (PMID:20147302, 27008021) version ‘2013-10-10-v2’, allowing maximum of two mismatches per 75 base sequence (parameters: ‘-M 2 -n 10 -B 2 -i 1 -N 1 -w 200000 -E 1 --pairmax-rna=200000 --clip-overlap’). Transcript annotation was based on the Gencode genes database (GENCODE 27, biomaRt2.48.3). To quantify gene expression levels, the number of reads mapping unambiguously to the exons of each gene was calculated. The Raw counts mapped to chromosome X, chromosome Y, mitochondrial genes, long non-coding RNA genes and genes with minimal expression (less than 3 counts in 95% of samples) were removed. To estimate the cell type fraction as well as cell type-specific gene expression in each sequenced sample, data were deconvoluted and B-cell specific gene expression was imputed using CIBERSORTxA standard Limma and Voom pipeline was applied to transform the raw counts to log2 counts per million (Log2-CPM). Fold-change, To calculate the transcriptomic distance between patients, shared nearest neighbour (SNN) patient clustering was conducted using the 