cells: spleen marginal zone B cells genotype: WT genetic background: C57BL/6J
Extracted molecule
total RNA
Extraction protocol
Antibody stained spleen cells were sorted using FACS Aria Cell-Sorting System (Beckton-Dickinson, Franklin Lake, NJ) and either 104 Marginal zone (MZ) cells or 104 follicular (FO) B cells were directly collected into RLT buffer (Qiagen, Valencia, CA). lysates were thawed in room temperature and genomic DNA was fragmented to by spinning the samples through Qiashredder columns for 2 minutes at 21,000 x g. To RLT buffer sample was added to 100% ethanol and RNAs were extracted using Qiagen RNeasy 96-well system as described previously (Virtaneva et al., 2005). As contaminating gDNA will interfere with the downstream Nugen Ribo-SPIA™ technology, each RNA sample were treated with 27 units of DNAse I (Qiagen, Valencia, CA) for 15 minutes at room temperature during extraction. RNA yield was estimated using Nanodrop (NanoDrop Technologies, Wilmington, DE) and 2100 Bioanalyzer (Agilent Technologies, Santa Clare, CA).
Label
biotin
Label protocol
Total of 5 µg of double stranded cDNA was concentrated to 25 µL. Each sample was combined with 5 µL fragmentation buffer mix (FL1) and 2 µL fragmentation enzyme mix (FL2). DNA was digested at 37 °C for 30 minutes and enzyme was denatured at 95 °C for 2 minutes. Fragmented ds cDNAs were biotin end-labeled using terminal transferase. Each fragmented sample was combined with15 μl labeling buffer mix (FL3), 1.5 μl biotinylated nucleotides (FL4), 1.5 μl terminal transferase (FL5), incubated at 37 °C for 60 minutes, and heat inactivated.
Hybridization protocol
Five micrograms of labeled target from twenty-four submitted samples (received 2-10-09) was combined with 2X hybridization buffer, 3nM B2 control oligo (Affymetrix 900457), 20X spike in (Affymetrix 51214), and DMSO making a final volume of 100ul for the twenty-four individual hybridizations to the commercial Affymetrix GeneChip, mouse gene 1.0 ST containing the Mus musculus genome. The hybridization cocktail, including the components listed above, was denatured for five minutes at 99◦C then transferred to a 45◦C heat block for an additional five minutes before transferring 80ul of the cocktail into the chip. The hybridization was carried out at a constant temperature of 45◦C for approximately seventeen hours using the Affymetrix 640 oven 500k approved.
Scan protocol
Upon completion of the fluidics process, each sample was scanned using the Affymetrix GeneChip 3000 7Gplus scanner to create the image files (dat). GeneChip Operating Software (GCOS v1.4) was then used to convert the image files to cel files
Description
Gene expression data spleen marginal zone B in WT A combination of CD23, CD21 and CD19 markers was included in the sorting scheme. PE-labeled anti-CD23 Ab was used to give a satisfying separation of MZ and FO B cells. MZ B cells were sorted as B220+CD21BriCD23-. FO B cells were sorted as B220+CD21loCD23+
Data processing
Data were collected using GeneChip Operating Software (GCOS v1.4). The robust multiarray average (RMA) and quantile normalization were performed.
