HD2-Bcell-BCR_IGH_IGK_IGL
Homo sapiens
Protocols: Peripheral blood was obtained from the Neuroinfection Department of the Research Centre of Neurology, Moscow, Russia. Venous blood was collected in EDTA Vacutainers (BD) from 9 MS patients (4 - benign MS, 5 - highly active MS) and 6 healthy donors (HD). The MS patients were between 23 and 83 years of age. Their scores on the EDSS ranged from 1.5 to 8.5. The EDSS values, scored on a scale of 0 to 10, were calculated with the Kurtzke EDSS scale. Blood samples were diluted two times in PBS with 2 mM EDTA and layered onto Ficoll-Paque Plus (GE Healthcare) and then centrifuged at 900 g for 40 minutes at room temperature. Isolated PBMCs from MS patients were incubated with ACK lysing buffer for complete deletion of any included red blood cells. Total B-cell pool and regulatory CD19+CD24highCD38high subpopulation were sorted individually for each individuum in tubes containing Qiazol lysis reagent (Qiagen, Germany). RNA extraction was performed using RNeasy Mini Kit (Qiagen, Germany) according to manufacturer's protocol. Reverse transcription (RT) was run as a 20 µL reaction with MMLV RT according to the manufacturer's instructions (Evrogen, Russian Federation). Degenerate primers for amplification of the variable fragments of the immunoglobulin repertoire from cDNA were modified from those previously described [Cheng, J. et al. PNAS, 2011]. Fifteen forward VH primers were used separately in each sample with a mixture of equal moles of four reverse JH primers. Thirteen Vκ primers and sixteen Vλ primers were separately used to amplify the VL genes with corresponding mixture of equal moles of two reverse Vκ or three reverse Vλ primers for each sample. . Library fragments of ~350 bp (variable fragment of IgH, IgK and IgL) were band isolated from an agarose gel. About 1 μg of the PCR product was ligated with adapters from NEBNext Multiplex Oligos using NEBNext Ultra DNA Library Prep Kit for Illumina (NEB).
ENA-FIRST-PUBLIC
2020-09-24T04:04:50Z
ENA-LAST-UPDATE
2020-09-14T15:53:28Z
External Id
SAMEA7306579
INSDC center name
Senior Research Fellow
INSDC first public
2020-09-24T04:04:50Z
INSDC last update
2020-09-14T15:53:28Z
INSDC status
public
Submitter Id
E-MTAB-9573:HD2-Bcell-BCR_IGH_IGK_IGL
age
40
broker name
ArrayExpress
cell type
B cell
clinical information
BCR chain: IGH,IGK,IGL
common name
human
developmental stage
adult
disease
normal
organism part
blood
sample name
E-MTAB-9573:HD2-Bcell-BCR_IGH_IGK_IGL
scientific_name
Homo sapiens
sex
female