characteristics: ERC000011
characteristics: ENA-CHECKLIST
characteristics: 2022-12-08T16:28:15Z
characteristics: ENA-FIRST-PUBLIC
characteristics: 2022-12-08T16:28:15Z
characteristics: ENA-LAST-UPDATE
characteristics: SAMEA110683330
characteristics: External Id
characteristics: INSERM U1151-Institut Necker-Enfants malades
characteristics: INSDC center name
characteristics: 2022-12-08T16:28:15Z
characteristics: INSDC first public
characteristics: 2022-12-08T16:28:15Z
characteristics: INSDC last update
characteristics: public
characteristics: INSDC status
characteristics: ERS12780897
characteristics: SRA accession
characteristics: E-MTAB-12117:P03_DN_D7_alpha
characteristics: Submitter Id
characteristics: ArrayExpress
characteristics: broker name
characteristics: B cell
characteristics: cell_type
characteristics: human
characteristics: common name
characteristics: Protocols: PBMCs from blood of healthy donors that was collected before and after Pneumovax vaccination, were isolated by centrifugation on Ficoll-Paque PLUS (GE Healthcare) and were stained with the following anti-human antibodies: APC-H7 anti-CD3/CD14/CD16, V500 anti-CD19, APC anti-CD27, PerCPcy5.5 anti-CD38, PE-Cy7 anti-CD24, Pe-Cy7 anti-IgD and FITC-CD43. All the mAbs were from BD Biosciences, except the anti-CD24 mAb (SONY) and the anti-IgM mAb (Biolegend). Dead cells were excluded by Sytox Blue staining (Invitrogen). Lymphocytes were first gated based on morphology, before exclusion of doublets, dead cells and CD3/CD14/CD16-positive cells. The sorted fractions and their phenotypes were as following : - plasmacells or \PC\ (CD19+CD27++CD38++)- \MZB\ cells (CD19+CD43-CD38lowCD27+IgM+IgD+)-switched memory cells or \SM\ (that encompasses switched and IgM-only cells (CD19+CD43-CD38lowCD27+IgD-)- double negative or \DN\ (CD19+CD43-CD38lowCD27-IgD-)-\naives\ B cells (CD19+CD43-CD38lowCD27-IgM+IgD+ The B cell fractions were sorted using a FACSAria III cell sorter (BD Biosciences) and were collected in Eppendorf tubes filled with PBS+ 2% fetal calf serum. After a centrifugation step and supernatant removal, the cell pellets were resuspended into 75 μl of RT lysis buffer (RNeasy Kit, Qiagen) and either frozen at -80°C or directly subjected to RNA extraction RNA extraction of the sorted cell fractions was done with the mini- or micro-RNeasy kit, according to the manufacturer’s instructions. Starting from a maximum of 150 ng of total RNA, mRNAs were then isolated with Dynabeads oligo (dT) from Thermo Fischer Scientific, according to the manufacturer’s guidelines 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 was synthetized in 10 μl (50°C for 1 hour and 72°C for 10 min) using SuperScript III Enzyme (Thermo Fisher Scientific). After ribonuclease H treatment (Clontech Takara) second-strand synthesis was performed in solid phase in 10 μl using Q5 Polymerase (New England Biolabs) and a mix of 13 primers covering IGHV leader sequences, containing 13 to 16 random nucleotide (UMI) and partial Illumina adaptor sequences (37°C for 20 min, 98°C for 30 s, 62°C for 2 min, and 72°C for 10 min). IGHV-D-J-CH double-stranded cDNA attached to the magnetic Dynabeads was washed three times in 10 mM Tris-HCl (pH 8.0) to remove the remaining primers, and the entire sample was used as a 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 (μ, α, and γ) primers (98°C for 30 s; 10 cycles of 98°C for 10 s, 58°C for 15 s, and 72°C for 1 min; 72°C for 10 min). Depending on the initial cell fraction, individual semi-nested PCR2 were performed with inner reverse primers specific for the mu, alpha, or gamma constant region and allowing the introduction of partial Illumina adaptors. Two microliters of the PCR1 product was used for the semi-nested PCR2 reactions carried in 20 μl (98°C for 30 s; 15 cycles of 98°C for 10 s, 58°C for 15 s, and 72°C for >1 min; 72°C for 10 min). The PCR2 products were purified with AMPure I XP beads at a ratio of 1:1, and 1 to 10 ng was 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. Sequencing was performed with the Illumina MiSeq sequencer using the V3 kit (2 × 300) with 12 pmol of the final pool and 10% PhiX control to avoid low-complexity library issues.
characteristics: description
characteristics: adult
characteristics: developmental stage
characteristics: normal
characteristics: disease
characteristics: CD19+CD43-CD38lowCD27-IgM+IgD+
characteristics: immunophenotype
characteristics: P03
characteristics: individual
characteristics: Homo sapiens
characteristics: organism
characteristics: Homo sapiens
characteristics: organism
characteristics: blood
characteristics: organism part
characteristics: Homo sapiens
characteristics: scientific_name
characteristics: male
characteristics: sex
characteristics: P03_DN_D7_alpha
characteristics: title