NextSeq 500 paired end sequencing; ScRNA-seq analysis of naive, germinal center and memory B cells of mice immunized with homologous and heterologous prime:boost combinations of whole-cell pertussis or acellular pertussis vaccines
ScRNA-seq analysis of naive, germinal center and memory B cells of mice immunized with homologous and heterologous prime:boost combinations of whole-cell pertussis or acellular pertussis vaccines
"B220+EYFP+PNA+GL7+ GC B cells, B220+EYFP+PNA-GL7- memory B cells and B220+EYFP-IgD+ and from dLNs of aP:wP, wP:aP and wP:wP vaccinated mice were first enriched by cell sorting, then viable GC and memory B cells were single cell FACS-sorted (AriaIII) into 384-well capture plates (Single Cell Discoveries) containing 384 primers and Mineral oil (Sigma) and immediately stored at -80°C. aP:aP dLN cells were first FACS enriched as B220+EYFP+ cells, due to their lower cell frequency, then B220+EYFP+PNA+GL7+ GC and B220+EYFP+PNA-GL7- memory B cells were single-cell sorted. The “Well id”, \""Cell Type\"", “Stimulus” attributes in plate layouts recapitulate all sorting information for each cell. This dataset was performed in three distinct batches: batch 1 [Samples: V01 to V05], batch 2 [Samples: V06 to V09], and batch 3 [Samples V10 to V12]. Each sample corresponds to a sorted 384 plate containing cells from one or multiple mice. A layout for each sample was added to recapitulate all the information needed for the correct analysis, such as “cell.type”, “stimulus”, “day.sorted”, “mouse.id” but also information referring to the status of markers “INDEX_” used for the sort : GL7, PNA and surface markers for downstream analysis :  Immunoglobulin status for each sorted cell. All plates from each of the three batches, regardless of the sorting date, were processed at the same time for library preparation and sequencing." Mice were subcutaneously (s.c) injected at the base of the tail with 100 µl of aP and wP vaccine formulations, provided by Sanofi-Pasteur, Marcy l'Etoile (Lyon), which correspond to 1/5 of the human dose but do not correspond to commercial vaccines as they lack additional components like tetanus or diphtheria toxins. The aP vaccine is composed of 20 μg/ml of pertussis toxoid (PT), 6 μg/ml of pertactin (PRN), 10 μg/ml of fimbriae 2,3 (Fim2,3), 10 μg/ml of filamentous haemagglutinin (FHA) and 0.66 mg/ml alum; the wP vaccine is made of 30 OU/ml whole cell pertussis and 2.4 mg/ml alum.Doses of 10 mg/mouse Nolvadex (Tamoxifen; AstraZeneca) in 300 μl of 20% Clinoleic (Baxter) were administered by gavage at day 7 , 10 and 31 post boost injection.  Cells were heat-lysed at 65° C followed by cDNA synthesis After second-strand cDNA synthesis, all the barcoded material from one plate was pooled into one library and amplified using in vitro transcription (IVT).  Following amplification, library preparation was done following the CEL-Seq2 protocol (Hashimshony T, Senderovich N, Avital G, Klochendler A, de Leeuw Y, Anavy L, Gennert D, Li S, Livak KJ, Rozenblatt-Rosen O, Dor Y, Regev A, Yanai I. CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq. Genome Biol. 2016 Apr 28;17:77. doi: 10.1186/s13059-016-0938-8.) to prepare a cDNA library for sequencing using TruSeq small RNA primers (Illumina)