NextSeq 500 paired end sequencing; Impact of Hells loss-of-function on DNA methylation in B cells during humoral immune responses: kinetic comparison of Mb1-Hells knockout and control mice by single-cell RNA sequencing
Impact of Hells loss-of-function on DNA methylation in B cells during humoral immune responses: kinetic comparison of Mb1-Hells knockout and control mice by single-cell RNA sequencing
The impact of Hells loss-of-function on DNA methylation maintenance was quantified by enzymatic-methyl sequencing of sample from conditional knockout (Mb1-Cre+/WT HellsF/F) and littermate control mice (Mb1-CreWT/WT HellsF/F). Three time points were chosen to follow the kinetics: day 0 for naïve B cells collection, day 7 and day 14 post NP-CGG intraperitoneal immunization for germinal center B cells collection, and day 14 for memory B cells.  At each time point, 2 mice of each genotype were used. In details: B220+CD95hiGL7hi viable splenic germinal center B cells at day 7 and day 14 post NP-CGG immunization were single cell FACS-sorted (AriaIII) into 384-well capture plates (Single Cell Discoveries) containing a small 50 nl droplet of barcoded primers and 10 μl of mineral oil (Sigma M8410) and immediately stored at -80°C. As a control B220+IgM- IgG1+GL7- CD38+ memory B cells (day 14) and B220+CD21+CD23+IgD+ naïve B (from day 7 immunized mice) cells were also single cell FACS-sorted. Due to their low frequency, memory B cells were first FACS enriched as B220+IgM-IgG1+ cells (AriaII).  Each sample corresponds to a sorted 384 plate containing cells from two mice (one wild type and one knock-out). A layout for each sample was added to recapitulate all the information needed for the correct analysis, such as “cell. type”, “Mouse.id”, “days_post_immunization”, “Genotype” but also information referring to the status of markers “INDEX_” used for the sort and additional surface markers for downstream analysis. The samples were separated in two batches for library preparation and sequencing, one batch containing Samples 1,2,4,5,7,8,10&11 and a second batch containing samples 3,6,9 and 12.  Mice were immunized intraperiotneally (i.p.) with 100µg of NP-CGG (4-hydroxy-3-nitrophenylacetyl-Chicken Gamma Globulin, Biosearch Technologies) dissolved in PBS and adsorbed on Alhydrogel aluminium hydroxide following manufacturer's instructions (Invivogen). 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).