NextSeq 550 paired end sequencing; Single cell RNA-sequencing of PBMC derived B cells from human healthy donor
Single cell RNA-sequencing of PBMC derived B cells from human healthy donor
PBMCs were separated from freshly-collected whole blood by density gradient centrifugation and stained with the following monoclonal antibodies: CD19-BV421 (Biolegend), IgD-PerCP- Cy5.5, CD27-PE-Cy7, CD38-APC, CD10-PECF594, CD3-APC-Cy7, IgG-BV605 (BD Biosciences) and viability dye eFluor780 (eBioscience) for 30 min on ice and, following two washes, cells were resuspended in PBS with 1% HI bovine serum for single cell sorting. Following exclusion of non-viable/CD3+ cells, individual transitional (CD19+, IgD+, CD27-,CD10+), mature naïve (CD19+, IgD+, CD27-, CD10-), switched memory (CD19+, IgD-, CD27+, IgG+) and plasmablast (CD19+, IgD-, CD27++, CD38++) were sorted directly into 96- well PCR plates (Eppendorf) using a FACS ARIA III (BD Biosciences). PCR plates were stored at –80 C for subsequent analysis. polyA tail Single cell RNA sequencing was performed with the Smart-Seq2 protocol as described by Picelli et al. (Picelli, et al., 2013; Picelli, et al., 2014) with the following modifications. Cell lysis buffer was prepared by adding 1μL Rnase inhibitor (Clontech, Mountain View, CA) to 19μl Triton X- 100 solution (0.2% v/v). Cells were sorted directly into 96-well plates containing 1μL lysis buffer, 0.5μL dNTP (10mM) and 0.5μL oligo-dT primer at 5uM. Reverse-transcription and PCR amplification were performed as described (Picelli, et al., 2013) with the following exceptions: reactions were performed at half volumes, the IS PCR primer was reduced to a 50nM final concentration and the number of PCR cycles increased to 28. Sequencing libraries were prepared using the Nextera XT Library Preparation Kit (Illumina; San Diego, CA, USA) at one quarter of the recommend total volume