GSM7659035: PBMCs patient pair_1, active stage T-PLL; Homo sapiens; RNA-Seq
After sampling, peripheral blood mononuclear cells (PBMCs) were isolated via density gradient centrifugation (Histopaque, Sigma-Aldrich). Cryo-preserved PBMCs were transferred to the Cologne Center for Genomics (CCG, Cologne) and CeMM-Biomedical Sequencing Facility (BSF, Vienna) for sequencing. Removal of dead cells was accomplished using a Dead Cell Removal Kit (Miltenyi Biotec) or by FACS sorting of DAPI-negative cells. Single-cell suspensions were processed with the Chromium Next GEM Single Cell 3ʹ Kit v3.1 (10x Genomics) with dual indices according to the manufacturer's instructions, using either one reaction per sample or multiplexing two to three samples with the Cell Multiplexing technology option. In short, cells and the appropriate master mix were loaded on a Chromium Next GEM Chip G and processed on the Chromium Controller to generate Gel Bead-In-Emulsions (GEMs). After the creation of barcoded full-length cDNA by incubation with cell lysate, primers, and a reverse transcription master mix, GEMs were broken, and the pooled fractions were recovered. Silane magnetic beads were used to remove leftover biochemical reagents and primers from the post-GEM reaction mixture. Full-length, barcoded cDNA was then amplified by PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were used to optimize the cDNA amplicon. Library construction was performed, including end repair, A-tailing, adaptor ligation, and PCR. The final libraries were quantified (Qubit), pooled, and the pool was quantified using the Peqlab KAPA Library Quantification kit and the Applied Biosystems 7900HT Sequence Detection System.