title: Illumina MiSeq paired end sequencing; immunoglobulin-mRNA seq repertoire of different human B cell subsets sampled at different time points after pneumovax vaccination to identify the B cell subset responsible for the production of anti-polysaccharides antibodies
design: immunoglobulin-mRNA seq repertoire of different human B cell subsets sampled at different time points after pneumovax vaccination to identify the B cell subset responsible for the production of anti-polysaccharides antibodies
library_construction_protocol: 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.