Illumina NovaSeq 6000 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 bulk 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 bulk RNA sequencing.
The transcriptomic impact of Hells loss-of-function was quantified by RNA 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 kinetic: day 0 for naïve B cells collection, day 7 and day 14 post NP-CGG intraperitoneal immunization for germinal center B cells collection. At each time point, 4 mice of each genotype were used.  Live cells were FACS sorted (AriaIII) using surface markers as follows: B220+CD21+CD23+ for naïve 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 RNA extraction. 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) Total RNA were isolated using the RNeasy Kit (QIAGEN) including a DNAse treatment step. RNA integrity and concentration were assessed by capillary electrophoresis using Fragment Analyzer (Agilent Technologies). RNAseq libraries were prepared starting from 188.3ng, 139.8 or 29.3ng of total RNA (3 groups) using the Universal Plus mRNA-Seq kit (Nugen) as recommended by the manufacturer. With this kit, stranded RNAseq libraries can be prepared from 10 ng to 1µg of total RNA. Briefly, the mRNA were captured with polyA+ magnetic beads form the total RNA. The mRNA were fragmented chemically. Single strand and second strand cDNA were produced and then ligated to Illumina compatible adaptators with Unique Dual Index. The cDNA produced were PCR amplified after an initial test to evaluate the suitable number of PCR cycle to apply to each sample. To produce oriented or 'stranded' RNAseq libraries, a final step of strand selection was performed.