Homo sapiens
Protocols: Immediately after splenectomy, mononuclear cells were isolated from crushed pieces of splenic tissue by ficoll density-gradient centrifugation and stored in liquid nitrogen. After thawing, mononuclear cells were counted and stained with antibodies directed against surface markers. Splenic B cell subsets from three RTX relapse patients, including PC (CD19+CD27highCD38highCD24-), GC (CD19+CD38intCD24-IgD-CD20+), switched memory (CD19+CD38-CD24+CD27+IgD-) and naive (CD19+CD38lowCD27-IgD+) B cells, were sorted in PBS/2% FCS with a FACS Aria III cell sorter (BD Biosciences). Approximately 104 to 2x105 cells were obtained for each subset. Cells were washed with PBS, centrifuged, and resuspended in the appropriate volume of Lysis/Binding Buffer from the Dynabeads mRNA DIRECT Micro Kit (ThermoFisher). mRNA were isolated directly with dynabeads oligo (dT) and immediately used in their entirety for reverse transcription in solid phase, according to a protocol adapted from Vergani et al, Frontiers in Immunology 8 (2017) with modified NGS primers (see sup. Table 5 , doi: https://doi.org/10.1101/833343). cDNA were synthetized in 10 μl (50°C 1 h, 72°C 10 min) using SuperScript III Enzyme (ThermoFisher). After RNase H treatment, second-strand synthesis was performed in solid phase in 10 μl using Q5 Polymerase (NEB) and a mix of 13 primers covering IGHV leader sequences, containing 13 to 16 random nt and partial Illumina adaptor sequences (37°C 20 min, 98°C 30 s, 62°C 2 min, and 72°C 10 min). IGHV-D-J-CH double-stranded cDNA attached to the magnetic dynabeads were washed three times in 10 mM Tris-HCl pH 8.0 to remove the remaining primers, and the entire sample was used as template for the PCR1 amplification in 10 μl using Q5 Polymerase with a forward primer that is specific for the partial Illumina adaptor sequence and allowing its extension, and a mix of reverse isotype specific (mu, alpha, gamma) primers (98°C 30 s; 10 cycles of 98°C 10 s, 58°C 15 s, and 72°C 1 min; 72°C 10 min). Depending on the initial cell fraction, individual semi-nested PCR2 were performed with inner reverse primers specific for the mu, alpha, and gamma constant region and allowing the introduction of partial Illumina adaptors. Two µl of the PCR1 product were used for the semi-nested PCR2 reactions carried in 20 μl (98°C 30 s; 15 cycles of 98°C 10 s, 58°C 15 s, and 72°C 1 min; 72°C 10 min). The PCR2 products were purified with Ampure XP beads at a ratio of 1:1, and 1-10 ng were used to add Illumina Index with the Nextera XT kit (Illumina). After a final purification with Ampure XP beads (ratio 1 :1), the library obtained for each sample was diluted at a concentration of 2 nM, and pooled equimolarly into a final pool.
ENA first public
2021-01-18
ENA last update
2020-12-21
External Id
SAMEA7745424
INSDC center alias
INSERM1151 - INEM
INSDC center name
INSERM1151 - INEM
INSDC first public
2021-01-18T04:08:56Z
INSDC last update
2020-12-21T15:46:02Z
INSDC status
public
Submitter Id
E-MTAB-9953:HD11_PC_mu
age
21
broker name
ArrayExpress
cell type
B cell
common name
human
developmental stage
adult
disease
normal
individual
HD11
organism part
spleen
response to treatment
healthy donor
sample name
E-MTAB-9953:HD11_PC_mu
sex
female