GSM4561503: Diagnosis and Relapse samples; Homo sapiens; RNA-Seq
Heparinised blood was collected, and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll/Hypaque density-gradient centrifugation. At diagnosis, B cells were purified by negative selection against CD2, CD3, CD4, CD14, CD15, CD16, CD34, CD56, CD61, CD235a (Glycophorin A) and FceRIa was used. At relapse, no purification was performed. All cells were cryopreserved with 10% DMSO/FBS and stored liquid nitrogen until the time of analysis. The cryopreserved cells were thawed in a water bath at 37ᵒC and resuspended in warmed RPMI medium (Sigma). Cell concentration and viability was determined using a LUNA™ cell counter (Logos Biosytems) and their viability was quantified. Cell hashing was carried out prior to pooling and loading on the 10x Chromium instrument. One million cells from each sample were separately suspended in 100 µl cell staining buffer (BioLegend) and 5 µl of Human TruStain FcX™ Fc Blocking reagent (Biolegend) was added and the samples incubated at 4 ᵒC for 10'. Both samples were incubated with 1 µg of TotalSeq™-A0251 Hashtag 1 or  TotalSeq™-A0252 Hashtag 2 antibody for  diagnosis and relapse, respectively. Samples were then incubated at 4 ᵒC for 30'. Cells were then washed 3x with 1 mL cell staining buffer (Biolegend), resuspended in PBS at a concentration of 1'000 cells/µl and then filtered through 40 µm strainers to ensure single cell suspension. Cell concentration and viability were verified using the LUNA™ cell counter (Logos Biosytems) before the two samples were pooled at a 50:50 ratio. This pool was then loaded onto the Chromium instrument as detailed by the manufacture. Libraries were generated using the 10x Genomics Chromium platform (version 3). scRNA-seq
GEO Accession
GSM4561503