NextSeq 500 paired end sequencing; Interference of B-Cell and T-Cell Receptor Genes in Single-Cell RNA Sequencing Data
Interference of B-Cell and T-Cell Receptor Genes in Single-Cell RNA Sequencing Data
Bone marrow samples were collected mechanically using a cordless drill from the femoral head of patients undergoing hip-replacement surgery. Larger debris was removed through filtering. After centrifugation and washing, the samples were layered carefully over Ficoll-Hypaque (GE Healthcare). Following centrifugation, bone marrow mononuclear cells were harvested and washed in PBS, before being frozen in aliquots and stored at -150Â°C. CD24hiCD38hi early B-lineage cells were electronically sorted with a Sony SH800. Staining with Fixable Viability Dye eFluor(TM) 506 (Invitrogen) was used to exclude dead cells. Cell suspensions were loaded into a Chromium cell processing unit (10x Genomics, Pleasanton, CA, USA), generating gel bead-in emulsions (GEMs). 5' gene expression libraries were constructed according to standard 10x protocol, using the Chromium Single Cell V(D)J Reagent Kits (v1.1 chemistry, 10x Genomics) GEMs were used to construct sequencing libraries using the standard 10x protocol. Genomics Chromium Single Cell V(D)J Reagent Kits (5', chemistry 1.1).