Miltenyi b cll isolation kit

Primary CLL PBMCs were obtained from the Stem Cell and Xenograft Core at the University of Pennsylvania (Philadelphia, PA). B-CLL cells were isolated using the Miltenyi B-CLL Isolation Kit (Miltenyi Biotec, #130-103-466). A full list of CLL donors used can be found in Supplementary Table S2.

mRNA sequencing was performed on RNA extracted with Qiagen RNeasy mini kits (Qiagen, Hilden, Germany). Libraries were prepared using TruSeq RNA kits (Illumina, Hayward, CA, USA) and sequenced using Illumina HiSeq2500 (n = 17) and HiSeq4000 (n = 21). Raw data was aligned to the hg38 reference genome using HISAT2 and read counts were calculated using HTseq-count against the Ensembl GRCh38.v94 GTF.
miRNA sequencing was performed on total RNA extracted with Qiagen miRNeasy kits. Libraries were prepared using Illumina TruSeq small RNA library kits and sequenced using Illumina HiSeq4000 (n = 17) and an Illumina HiSeq2500 (n = 21). Confirmatory miRNA sequencing to preclude the contribution of contaminating T-cell/monocytes was processed as before, but from purified CLL cells (Miltenyi B-CLL Isolation kit (Miltenyi, Bergisch Gladbach, Germany)), and sequenced on an Illumina HiSeq 4000. miRNA data in the form of fastq were aligned to the hg38 reference genome using BWA v0.7.12 (RRID:SCR_010910) and read counts calculated using HTseq-Count against miRbase v21.