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GSC RT-PCR amplification of 10 cells (SP & CD8 T cells), single SP cell and single-SP-cell equivalent

ABSTRACT: GSM48315-GSM48332: Ten cells from C57Bl/6 male mouse bone marrow (SP or CD8 T cells) were sorted into individual wells of 96-well plates. The mRNA of these cells was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. GSM48333-GSM48344: Single SP cell from C57Bl/6 male mouse bone marrow were sorted into individual wells of 96-well plates. The mRNA of these single cells was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. GSM48345-GSM48349: Forty bone marrow SP cells from C57Bl/6 male mouse bone marrow, Sca-1 positive and Gr-1 negative, gated on the tip of the SP tail, were sorted into 160 microliters of lysis buffer (40 times the amount used for single cells). 4-microliter aliquots (containing the mRNA equivalent to one single cell) were dispensed into individual wells of 96-well plates. The mRNA contained in each aliquot was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. Detection of the microarray hybridization signals was done according to the standard Affymetrix protocol (antibody amplified). Keywords = HSC Keywords = stem cell Keywords = SP Keywords = side population Keywords = CD8 Keywords = lymphocytes Keywords = global single cell RT-PCR Keywords = GSC RT-PCR Keywords: parallel sample

ORGANISM(S): Mus musculus

PROVIDER: GSE2534 | GEO | 2006/01/02

SECONDARY ACCESSION(S): PRJNA92689

REPOSITORIES: GEO

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Transcription profiling and GSC RT-PCR of mouse bone marrow cells SP and CD8 T cells after amplification, single SP cell and single-SP-cell equivalent to examine diversity of single hematopoietic stem cells

Project description:GSM48315-GSM48332: Ten cells from C57Bl/6 male mouse bone marrow (SP or CD8 T cells) were sorted into individual wells of 96-well plates. The mRNA of these cells was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. GSM48333-GSM48344: Single SP cell from C57Bl/6 male mouse bone marrow were sorted into individual wells of 96-well plates. The mRNA of these single cells was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. GSM48345-GSM48349: Forty bone marrow SP cells from C57Bl/6 male mouse bone marrow, Sca-1 positive and Gr-1 negative, gated on the tip of the SP tail, were sorted into 160 microliters of lysis buffer (40 times the amount used for single cells). 4-microliter aliquots (containing the mRNA equivalent to one single cell) were dispensed into individual wells of 96-well plates. The mRNA contained in each aliquot was amplified by the global single cell RT-PCR method and biotinylated targets were generated after optimal digestion with DNAse I. Detection of the microarray hybridization signals was done according to the standard Affymetrix protocol (antibody amplified).

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RNA Sequencing Analysis of T Cell Receptor Alpha and Beta Chain in Thymic WT versus CIC-deficient CD4+ SP Non-Treg and Treg Cells.

Project description:Purpose : The goal of this study is to compare the repertoire of T cell receptor (TCR) alpha and beta chain between WT and CIC-deficient thymic CD4+ single positive (SP) T cells, in both non-Treg and Treg populations, to confirm the effect of CIC deficiency on T cell development in the aspect of TCR repertoire. Methods : Treg (CD4+CD8-CD25+GFP+) and non-Treg (CD4+CD8-GFP-) cells from Foxp3-GFP;Cicf/f and Foxp3-GFP;Cicf/f;Vav1-Cre mice were sorted by MoFlo-XDP (Beckman Coulter). Total RNA was extracted using RiboEX (GeneAll) according to the manufacturer's instructions. RNA was then amplified using a commercially available multiplex primer mix covering the TCR alpha and beta chains in two separate PCR reactions. Reverse transcription and subsequent PCR amplification (RT-PCR1) were performed using the Qiagen OneStep RT PCR mix (Qiagen). The cDNA was selected and unused primers were removed by SPRIselect bead selection (Beckman Coulter) followed by a second round of amplification performed with a pair of primers specific for communal sites engineered onto the 5ʹ end of the C- and V- primers used during RT-PCR1. After library preparation, paired-end sequencing was performed using the Illumina Miseq v3 600-cycle Reagent Kit (Illumina). Results : We observed increased frequency of TCRs with long CDR3 length in CIC-deficient non-Treg and Treg cells. We also found significant alterations in the TCR repertoire of TCR beta chain in CIC-deficient Treg cells compared to WT cells, including altered frequency of V and J chain usage. Conclusions : Our study represents the first comparative analysis of TCR repertoire of thymic CD4 SP cells from WT and hematopoietic lineage cell-specific Cic deficient mice. We concluded that CIC deficiency leads to alterations in TCR repertoire of thymic Treg cells.

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