Experiment Detail
| Title | Illumina HiSeq 2500 paired end sequencing; Intrinsic transcriptional heterogeneity in B cells controls early class switching to IgE |
| Design Description | Intrinsic transcriptional heterogeneity in B cells controls early class switching to IgE |
| Organism | Mus musculus |
Library Description
| Name | batch2_P5_neg_B10 |
| Strategy | RNA-Seq |
| Source | TRANSCRIPTOMIC |
| Selection | RT-PCR |
| Layout | PAIRED |
| Orientation | |
| Nominal Length | 350 |
| Nominal Sdev | 100 |
| 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. |
Platform
| Platform | ILLUMINA |
| Instrument Model | Illumina HiSeq 2500 |
Processing
| Base Calls | |
|---|---|
| Sequence Space | |
| Base Caller | |
Spot Information
| Number of Reads per Spots | 0 |
| Spot Length | 250 |
Read Spec
| Read Index 0 | |
|---|---|
| Read Label | |
| Read Class | Application Read |
| Read Type | Forward |
| Base Coord | 1 |
| Read Index 1 | |
| Read Label | |
| Read Class | Application Read |
| Read Type | Reverse |
| Base Coord | 126 |
Navigation
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Submission | ERA656423 |
 FTP
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Study | ERP016192 | ||
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Sample | ERS1216439 | ||
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Run | ERR1468005 |
FASTQ
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SRA
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