strain/background: C57BL/6J-ChrY^SJL gender: male age: 8 weeks tissue: lymph nodes cell type: CD4+ T cells
Extracted molecule
total RNA
Extraction protocol
Three biological replicates were pooled from the axillary, brachial, and inguinal lymph nodes from 5 mice for each replicate. Cells were washed with PBS/1%FCS, pelleted at 300g for 5 min at 4oC. A working stock of Live/Dead-775 dye (Invitrogen L10119) was made by adding 1ul of reconstituted dye to 1ml PBS and cells were resuspended in 100ul of working stock per 1x10^6 cells. Cells were incubated for 30 minutes on ice in the dark. Cells were washed with cold PBS/1%FCS, pelleted, and resuspended in 100ul of an antibody mixture containing 1ug/ml PerCp-Cy5.5 anti-mouse TCRβ chain and 1ug/ml Alexa Flour® 488 anti-mouse CD4 antibodies in PBS/1%FCS. Cells were incubated on ice in the dark for 30 minutes then washed with cold PBS/1%FCS, pelleted, and resuspended at a concentration of 10x106 cells per 1ml of PBS/10%FCS. Cells were filtered through 50um nylon mesh and sorted using BD FACSAria to collect ~3x10^6 CD4+ T cells per sample. RNA was isolated using the miVana miRNA isolation Kit following the manufacturer's instructions (Ambion).
Label
Biotin
Label protocol
Total RNA containing small RNA was labeled using the Flashtag RNA labeling kit (Genisphere, Hatfield, PA, USA) and performed in the Vermont Genetics Network Microarray Facility according to the manufacturer's instructions. Briefly, for each sample, 500ng of total RNA were subjected to a tailing reaction (2.5 mM MnCl2, ATP, Poly A Polymerase - incubation for 15 minutes at 37°) followed by ligation of the biotinylated signal molecule to the target RNA sample (1× Flash Tag ligation mix biotin, T4 DNA ligase - incubation for 30 minutes at 25oC) and addition of HSR stop solution.
Hybridization protocol
Each sample was hybridized to a GeneChip® miRNA 2.0 Array (Affymetrix, Santa Clara, CA, USA) at 48°C and 60 rpm for 16 hours then washed and stained on Fluidics Station 450 (Fluidics script FS450_0003).
Scan protocol
After staining, arrays were scanned (3000-7G Scanner, Affymetrix Inc., Santa Clara, CA) and data collected for statistical analysis.
