NextSeq 500 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 enzymatic-methyl 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 enzymatic-methyl sequencing
Mice were immunized intraperitoneally (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). 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 naive B cells collection, day 7 and day 14 post NP-CGG intraperitoneal immunization for germinal center B cells collection. At each time point, 2 mice of each genotype were used. Live cells were FACS sorted (AriaIII) using surface markers as follows: B220+CD21+CD23+ for naive B cells, and B220+CD95+GL7+ for germinal center B cells. Due to their low frequency, germinal center B cells were FACS-sorted twice using the same gating strategy. Collected samples were lysed and processed for DNA extraction. DNA extraction from germinal centre B cells for EM-Seq was performed using the Kit Quick-DNA Microprep Plus (Zymo). DNA library construction protocol for EM-Seq, performed by EMBL GeneCore. The library was prepared according to NEBnext Enzymatic Methyl-seq kit (New England BioLabs, E7120).