characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-07-26T16:22:24Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-07-26T16:22:24Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA112354190
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: 2023-07-26T16:22:24Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-07-26T16:22:24Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS14465088
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-12499:Sample 2
characteristics.characteristic: Submitter Id
characteristics.text: 20
characteristics.unit: week
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.text: mixed
characteristics.characteristic: cell_type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: 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).
characteristics.characteristic: description
characteristics.text: adult
characteristics.characteristic: developmental stage
characteristics.text: wild type genotype and Mb1-Hells knockout
characteristics.characteristic: genotype
characteristics.text: 861 and 862
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: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.characteristic: sex
characteristics.text: C57BL/6J
characteristics.characteristic: strain
characteristics.text: Sample 2
characteristics.characteristic: title