title: Single-cell RNA sequencing analysis of circulating leucocytes and bone marrow cells from a patient with HYOU1 Pro444His variant and heatlhy controls
design: Single-cell RNA sequencing analysis of circulating leucocytes and bone marrow cells from a patient with HYOU1 Pro444His variant and heatlhy controls
library_construction_protocol: Blood cells from standard venopuncture were treated with red blood cell lysis solution 1X (Miltenyi), washed and resuspended in PBS containing 2% fetal bovine serum to a concentration of ~800-1,000 cells/µl with high viability >95%.  The single-cell suspension was loaded at a volume to target of around 5000 cells per sample onto a Chromium Controller (10x Genomics, Pleasanton, CA) for Gel Bead-In-EMulsions “GEM” generation according to manufacturer's instructions (10X Genomics, Pleasanton, CA). After GEM generation, single index libraries were generated using the Chromium Single Cell 3' Library and Gel Bead kit v3.1, according to manufacturer's instructions (PN 1000121, 10X Genomics, Pleasanton, CA). Size distribution and concentration of cDNA and final libraries were verified on an Agilent Bioanalyzer High Sensitivity chip (Agilent Technologies, Santa Clara, CA) and the concentration of the libraries were measured using Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA) and Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA).