Single-cell RNA-seq library preparation using 10x Genomics

Frozen BM cells were rapidly thawed, washed, counted and resuspended in PBS and 0.04% BSA to a final concentration of 1000 cells/μl. The Chromium^™ Controller (10x Genomics, Pleasanton, CA, USA) was used for parallel sample partitioning and molecular barcoding^{61 (link)}. To generate a single-cell Gel bead in EMulsion (GEMs), cellular suspensions were loaded on a Single Cell 3’ chips together with the Single cell 3’ Gel Beads, according to the manufacturer’s instructions (10x Genomics). Single-cell RNA-Seq libraries were prepared using ChromiumTM Single Cell 3’ Library Kit v2 (10x Genomics). 14 cycles were used for the total cDNA amplification reaction and for the total sample index PCR. Generated libraries were combined according to Illumina specifications and paired-end sequenced on HiSeq 2500–4000 platforms with standard Illumina sequencing primers for both, sequencing and index reads. 100 cycles were used for sequencing Read1 and Read2.