title: Illumina HiSeq 2500 paired end sequencing; Intrinsic transcriptional heterogeneity in B cells controls early class switching to IgE
design: Intrinsic transcriptional heterogeneity in B cells controls early class switching to IgE
library_construction_protocol: 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: Experimental Factor: eGFP positive
attributes.value: phenotype
attributes.tag: Experimental Factor: batch1_GFP_G05
attributes.value: single cell ID
attributes.tag: Experimental Factor: batch 1
attributes.value: block