strain: B6 cell type: spleen-derived B cells genotype: Wild type age (months): 8 developmental stage: adult sex: Female
Treatment protocol
None.
Growth protocol
Six Nf1+/- B6 (three males, three females) and six wild-type (three males, three females) adult (“Nf1-Mouse-12”) mice from 5 litters of the same line were euthanized at the age of ~8 months with CO2. The dissected spleens were homogenized with the tip of a syringe plunger (3 ml syringe, slip tip, BD Biosciences, Discovery Labware, Bedford, MA, USA) and passed through a 70 μm nylon cell strainer (BD Biosciences) and purified using the CD19 mouse microbeads (Miltenyi Biotec, Auburn, CA, USA) on the autoMACS Separator (Miltenyi Biotec) using the possel_d program, following the manufacturer’s directions. CD19+ and CD19- flow-through fractions were collected, and the purity was assessed by FACS on aliquots using GR1-FITC, B220-PE, CD3-PerCPCy5.5 and MAC1-APC antibodies (BD Pharmingen, San Diego, CA, USA). The remaining CD19+ and CD19- fractions were immediately lysed in 1 mL of the Trizol reagent (Invitrogen) and frozen at -80oC.
Extracted molecule
total RNA
Extraction protocol
Trizol cell lysates were mixed with chloroform (1/5 of lysate volume), vortexed for one minute and centrifuged in a table-top centrifuge at 13,000 rpm for 15 min at 4ºC. The aqueous phase was used for RNA isolation. The aqueous phase was mixed with equal volume of 70% ethanol and immediately loaded onto RNeasy mini columns (Qiagen, Valencia, CA, USA), with subsequent steps performed as per the manufacturer’s protocol. The RNA quality was estimated on a 2100 Bioanalyzer, RNA 6000 Nano Chips (Agilent, Santa Clara, CA, USA). Samples with RNA integrity number (RIN) of 8.0 and above were used for further analysis.
Label
Cy3
Label protocol
Biotinylated cRNA were prepared with the Illumina TotalPrep RNA amplification kit (Ambion Inc., Austin, TX) per the manufacturer's instuctions.
Hybridization protocol
Amplified biotinylated cRNA (1.5 ug) were hybridized to Mouse Ref-6_v1 Sentrix BeadChips. In all steps, care was exercised to avoid batch effects. Samples were hybridized to the microarrays at 55ºC for 16-17 hours. Microarrays were washed to remove non-specifically bound cRNA, stained with 1 mg/mL Streptavidin-Cy3 (GE Healthcare, Piscataway, NJ, USA) and dried.
Scan protocol
Microarrays were scanned in BeadStation 500 scanner. Image acquisition and initial image analysis were done with Illumina BeadScan and BeadStudio applications.
Description
12-wt
Data processing
Raw data from the Illumina BeadChips was corrected for background and quantile normalized using BeadExplorer (version 1.5.0), a Bioconductor module developed for quality control, normalization, annotation and exploration of Illumina BeadChip data. Average detection value was calculated for each transcript, and the transcripts with average detection values below 0.99 were excluded from further analysis.
Non-normalized data is available in the 'GSE18447_non-normalized_data.txt' file, which is linked to the Series GSE18447 record as a supplementary file.