RNA-seq of human atypical B cell subset 6: donor 3418
RNA-seq libraries were generated following the NEBNext Single Cell/Low Input RNA Library Prep Kit for Illumina (NEB #E6420S) protocol for cells, except that all DNA cleanup steps we done using SparQ magnetic beads (Quantabio). In brief, 200 cells of each B cell subset were FACS sorted directly into 1 NEBNext Cell Lysis Buffer in 1.7 ml low binding tubes (Corning #3207). Cells were then flash frozen in liquid nitrogen for future use. For all samples, cDNA was synthesized and amplified using 21 cycles. cDNA quantity and quality were assessed by Invitrogen Qubit and Agilent 2100 Bioanalyzer. PCR enrichment of adaptor-ligated DNA was performed using NEBNext Multiplex Oligos for Ilumina (NEB #E7335S and #E7500S) following the recommended number of amplification cycles based on the amount of cDNA input used in the fragmentation/end prep reaction. Library quantity and quality were assessed by Invitrogen Qubit and Agilent 2100 Bioanalyzer.