title: batch1_Tom_C01
organism: Mus musculus
description: Protocols: Splenic B cells were isolated from mice aged 9-16wks by depletion of CD43+ cells 65 using magnetic microbeads (Miltenyi Biotec, Cat no. 130-049-801). Purity of the isolated B cells was assessed using anti-B220 antibody (clone RA3-6B2) and found to be >98% for B220+ cells. To induce class-switching to IgG1 and IgE, B cells were seeded into 96-well round bottom plates at a density of 105 cells/200 μl RPMI supplemented with 10% FBS, 50 μM 2-mercaptoethanol, 100 U/ml penicillin- 100 μg/ml streptomycin (Invitrogen; Cat no. 15140122), 25 ng/ml recombinant mouse IL-4 (R&D Systems; Cat no. 404-ML), 40 μg/ml LPS (Sigma-Aldrich; L4391 or L3129) and 20 ng/ml recombinant mouse BAFF of (R&DSystems; Cat no. 2106-BF). Cells were harvested after 24-hours for FACS as detailed in the cell-lysis protocol\. After treatment as described in the stimulation protocol, single cells were deposited into 96-well PCR plates containing lysis buffer using an iCyt Synergy cell sorter. Plates were then immediately spun down and stored at -80°C until further processing. The generation of the single-cell libraries followed the Smart-seq2 protocol42, then switched to the Fluidigm C1 protocol for tagmentation and purification of the pooled libraries (Fluidigm; PN 100-7168). The pooled, barcoded libraries were quantified by qPCR and checked for size distribution using an Agilent Bioanalyzer 2100.
attributes.tag: ENA first public
attributes.value: 2016-12-12
attributes.tag: ENA last update
attributes.value: 2016-06-22
attributes.tag: External Id
attributes.value: SAMEA4045191
attributes.tag: INSDC center alias
attributes.value: Wellcome Trust Sanger Institute
attributes.tag: INSDC center name
attributes.value: Wellcome Trust Sanger Institute
attributes.tag: INSDC first public
attributes.value: 2016-12-12T11:05:34Z
attributes.tag: INSDC last update
attributes.value: 2016-06-22T08:53:28Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-4825_2:batch1_Tom_C01
attributes.tag: age
attributes.value: 9 to 16
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: lymphocyte of B lineage
attributes.tag: common name
attributes.value: house mouse
attributes.tag: genotype
attributes.value: I1-eGFP x I-tdTom
attributes.tag: phenotype
attributes.value: tdTomato positive
attributes.tag: sample name
attributes.value: E-MTAB-4825_2:batch1_Tom_C01
attributes.tag: sex
attributes.value: male
attributes.tag: stimulus
attributes.value: Interleukin 4, lipopolysaccharide, B cell activating factor
attributes.tag: strain
attributes.value: BALB/c