characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-07-26T16:22:27Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-07-26T16:22:27Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112647959
characteristics.characteristic: External Id
characteristics.text: Institut Necker-Enfants Malades, INSERM U1151/CNRS UMR 8253, Universite Paris Descartes, Sorbonne Paris Cite, 75993 Paris Cedex 14, France.
characteristics.characteristic: INSDC center name
characteristics.text: 2023-07-26T16:22:27Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-07-26T16:22:27Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14645682
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12638:D0 - KO3
characteristics.characteristic: Submitter Id
characteristics.text: 14
characteristics.unit: week
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: naive B cell
characteristics.characteristic: cell_type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: 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.
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: HellsF/F Mb1Cre+
characteristics.characteristic: genotype
characteristics.text: 340 - 8
characteristics.characteristic: individual
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: spleen
characteristics.characteristic: organism part
characteristics.text: Mb1 Cre-driven knockout of Hells
characteristics.characteristic: phenotype
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.characteristic: sex
characteristics.text: C57BL/6J x 129Sv
characteristics.characteristic: strain
characteristics.text: D0 - KO3
characteristics.characteristic: title