Protocols: Venous blood was collected in EDTA Vacutainers (BD). Blood samples were diluted two times in PBS with 2 mM EDTA and layered onto Ficoll (Paneco) and then centrifuged at 300 g for 30 minutes at room temperature. Cell were separated with fluorescence activated cell sorting (BD FacsAria III, BD Biosciences) into the following subpopulations: memory B-cells (Bmem, CD19+ CD20+ CD27+ CD138-), plasmablasts (PBL, CD20- CD19+ CD27++ CD138-), plasma cells (PL, CD20- CD19 Low/- CD27+ CD138+) individually for each donor directly into RLT buffer (Qiagen, Germany). RNA extraction was performed using RNeasy Mini Kit (Qiagen, Germany) according to manufacturer's protocol Immunoglobulin heavy chain (IGH) cDNA libraries were prepared according to the protocol described earlier (Turchaninova et al. 2016) with several modifications. Briefly, we used 5'-RACE (Rapid Amplification of cDNA Ends) approach based on a template-switch effect to introduce 5' adaptors during cDNA synthesis. These adaptors contain both unique molecular identifiers (UMIs), allowing error-correction, and sample barcodes (see Zvyagin et al. 2017 for the adaptor structure) for ruling out cross-sample contaminations. Universal sequence for annealing of forward PCR primer was also introduced by the adaptor. The set of primers specific for C-segment was used in an multiplex RT reaction followed by PCR amplification step with another semi-nested set of C-segment specific primers preserving isotype information.
ENA first public
2022-01-01
ENA last update
2022-01-01
External Id
SAMEA11308693
INSDC center alias
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry
INSDC center name
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry
INSDC first public
2022-01-01T00:13:21Z
INSDC last update
2022-01-01T00:13:21Z
INSDC status
public
Submitter Id
E-MTAB-11193:Sample 37
age
39
broker name
ArrayExpress
cell type
plasmablast
common name
human
developmental stage
adult
disease
normal
individual
IM
organism part
peripheral blood
replicate label
3
sample name
E-MTAB-11193:Sample 37
sex
male
time
p02