Unknown,Transcriptomics,Genomics,Proteomics

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RNA seq of rectal tumour tissue and paired healthy tissue, with EpCAM purification

ABSTRACT: Tumour tissue was immediately obtained post-surgery along with paired healthy tissue near (<2cm from tumour margin) and far (>10cm) from the tumour site. Each tissue was dissociated and stained with flow cytometry antibodies and sorted by FACS into Live/Dead-negative EpCAM-positive CD3-negative populations. An additional sample was passed through the FACS however no sorting was applied (control for the effect of sorting). Total RNA was purified and tissues compared for gene expression to identify potential hits that were assessed for protein expression and immunogenicity in later experiments.

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Homo sapiens

SUBMITTER: Alex Greenshields-Watson

PROVIDER: E-MTAB-8803 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Memory B cells play essential roles in the maintenance of long-term immunity and may be important in the pathogenesis of autoimmune disease, but how these cells are distinguished from their naÃ¯ve precursors is poorly understood. To address this, it would be important to understand how gene expression differs between memory and naive B cells in order to elucidate memory-specific functions. Using model systems that help overcome the lack of murine memory-specific markers and the low frequency of antigen-specific memory and naÃ¯ve cells, we undertook a global comparison of gene expression between memory B cells and their naive precursors. 1st generation screen:; These data represent our first generation comparison of gene expression between murine naÃ¯ve and memory splenic B cells. NaÃ¯ve NP-binding splenic B cells were FACS purified from unimmunized mVh186.2 transgenic Balb/c mice. Memory B cells were generated by immunizing mVh186.2 transgenic Balb/c mice with 2 doses of NP-CGG in alum delivered i.p. 6 weeks apart. After a minimum of 12-weeks rest, NP-binding splenic B cells were isolated by FACS. Total RNA was extracted, cRNA synthesized and labeled and hybridized to Affymetrix mouse 430 2.0 chips. 2nd generation screen:; These data represent our second generation comparison of gene expression between murine naÃ¯ve and memory splenic B cells. NaÃ¯ve NP-binding AA4.1neg splenic B cells were FACS purified from unimmunized mVh186.2 transgenic Jk KO Balb/c mice. Memory B cells were generated from these naive precursors after adoptive transfer into recipients that mount poor endogenous NP-responses. 12-weeks post i.p. immunization with NP-CGG in alum, NP-specific splenic memory B cells were isolated by FACS. Total RNA was extracted, cRNA synthesized and labeled and hybridized to Affymetrix mouse 430 2.0 chips. Memory/NaÃ¯ve comparison data linked below as Supplementary files. SERIES_CITATION: Tomayko, MM, Anderson, SM, Brayton CE, Sadanand, S, Steinel NC, Behrens, TW, Shlomchik, MJ. 2008. Systematic comparison of gene expression between murine memory and naÃ¯ve B cells demonstrates that memory B cells have unique signaling capabilities. J. Immunol., in press, 2008. Experiment Overall Design: 1st generation screen: 7 samples, 3-4 biological replicates each of murine splenic naÃ¯ve and memory B cells Experiment Overall Design: 2nd generation screen: 8 samples, 4 biological replicates each of murine splenic naÃ¯ve and memory B cells

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RNA seq of rectal tumour tissue and paired healthy tissue, with EpCAM purification

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