Project description:The indicated thymic progenitor population was sorted via FACS and then loaded into a Fluidigm C1 small cell capture chip for single-cell capture, lysis, reverse transcription, and preamplification. Preamplified products were analyzed on a BioMark HD with EvaGreen chemistry. The genes analyzed were selected based on bulk RNA sequencing data and/or prior publications, including genes relevant for gamma/delta T cells and T cell progenitors. B6=C57BL/6J, Rag=B6.Rag1-/-, Tcrd=B6.Tcrd-/-, DN1d=Live/TCRd-/Lin-/CD44+/CD25-/CD24+/cKit-, DN2=Live/TCRd-/Lin-/CD44+/CD25+/cKit+, cKit-=Live/TCRd-/Lin-/CD44+/CD25-/cKit-. Lin=CD3/CD8/CD11b/CD11c/CD19/Gr-1/NK1.1/TCRb/Ter-119 Sample naming nomenclature is as follows: MouseLine_CellPopoulation_DateCaptured_CaptureChamber_CellNumberInChamber
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). 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
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).
Project description:Three HCT116 cell lines (HCT WT, HCT p21-/-, HCT p53-/-) were analyzed by RT-qPCR array analysis for chromatin modification enzymes to identify potential target genes, that depend on the cell cycle regulator p21 and that are indpendent of p53. HCT116 cell lines (HCT WT, HCT p21-/-, HCT p53-/-) were cultured at 37°C in a humidified atmosphere with 5% CO2. Cells were harvested at a confluency of ~60-80% and RNA was isolatd using QIAzol an Rneasy Kits from Qiagen. cDNA was produced from equal amounts of RNA using the RT² First Strand Ki from Qiagen and RT-qPCR analysis was performed following the manufacturer's instructions using the Epigenetic Chromatin Modification Enzymes RT² Profiler PCR Array from Qiagen. The CFX96TM Real-Time System and the C1000TM Thermal Cycler from Bio-Rad were used to perform the RT-PCR runs. The three different cell lines were analyzed in three biological triplicates. The mean expression Ct values of the target genes were normalized against the mean Ct value for GAPDH to calculate the Fold Change values. The Raw Data was processed using the RT² Profiler PCR Array Data Analysis version 3.5 software from SABiosciences.